Genetically modified mice comprising humanized cellular immune system components with improved diversity of tcrb repertoire

ABSTRACT

Disclosed herein are non-human animals (e.g., rodents, e.g., mice or rats) genetically engineered to express a humanized T cell co-receptor (e.g., humanized CD4 and/or CD8 (e.g., CD8α and/or CD8β)), a human or humanized T cell receptor (TCR) comprising a variable domain encoded by at least one human TCR variable region gene segment and/or a human or humanized major histocompatibility complex that binds the humanized T cell co-receptor (e.g., human or humanized MHC II (e.g., MHC II α and/or MHC II β chains) and/or MHC I (e.g., MHC Iα) respectively, and optionally human or humanized β2 microglobulin). Also provided are embryos, tissues, and cells expressing the same. Methods for making a genetically engineered animal that expresses at least one humanized T cell co-receptor (e.g., humanized CD4 and/or CD8), at least one humanized MHC that associates with the humanized T cell co-receptor (e.g., humanized MHC II and/or MHC I, respectively) and/or the humanized TCR are also provided. Methods for using the genetically engineered animals that mount a substantially humanized T cell immune response for developing human therapeutics are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119 (e) to U.S. Provisional Patent Application Ser. No. 63/168,774, filed Mar. 31, 2021, the disclosure of which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 10948US01_ST25.txt, created on Mar. 30, 2022, and having a size of 60 kilobytes, and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to a non-human animals (e.g., rodents, e.g., mice or rats) capable of mounting substantially human(ized) T cell mediated immune responses and expressing (i) one or more human(ized) T cell co-receptor(s) (e.g., CD4 and/or CD8 (e.g., CD8α, and/or CD8β)), (ii) one or more human(ized) major histocompatibility complex(es) that associates with the one or more human(ized) T cell co-receptor(s) (e.g., MHC II (e.g., MHC α and/or MHC II β) and/or MHC I (e.g., MHC I α and/or β2 microglobulin)) and/or (iii) a human(ized) T cell receptor (TCR) (e.g., TCRα and/or TCRβ); embryos, tissues, cells and/or nucleic acids isolated from the non-human animals; methods of making the non-human animals; and methods of using the non-human animals for the development of human therapeutics.

BACKGROUND OF THE INVENTION

In the adaptive immune response, foreign antigens are recognized by receptor molecules on B lymphocytes (e.g., immunoglobulins) and T lymphocytes (e.g., T cell receptor also referred to as TCR). These foreign antigens are presented on the surface of cells as peptide fragments by specialized proteins, generically referred to as major histocompatibility complex (MHC) molecules, and specifically referred to as human leukocyte antigen (HLA) in humans. During a T cell-mediated response, antigens presented by MHC molecules are recognized by a T cell receptor. However, more than T cell receptor recognition of MHC-antigen complex is required for an effective immune response. The binding of a T cell co-receptor molecule (e.g., CD4 or CD8) to an invariant portion of MHC is also required.

T cells come in several varieties, including helper T cells and cytotoxic T cells. Helper T cells typically express co-receptor CD4 and recognize antigens bound to MHC II molecules. CD4+ T cells activate other effector cells in the immune system, e.g., MHC II expressing B cells to produce antibody, MHC II expressing macrophages to destroy pathogens, etc. The binding of CD4 and T cell receptor to the same MHC II-presented foreign antigen makes a T cell significantly more sensitive to that antigen.

In contrast, cytotoxic T cells (CTLs) typically express co-receptor CD8 and recognize foreign antigens bound to MHC I molecules. CTLs are specialized to kill any cell that bears an MHC I-bound peptide recognized by its own membrane-bound TCR. When a cell displays peptides derived from cellular proteins not normally present (e.g., of viral, tumor, or other non-self origin), such peptides are recognized by CTLs, which become activated and kill the cell displaying the peptide. Similar to CD4, engagement of CD8 makes CTLs more sensitive to MHC I-presented antigen.

Not all antigens will provoke T cell activation due to tolerance mechanisms. However, in some diseases (e.g., cancer, autoimmune diseases) peptides derived from self-proteins become the target of the cellular component of the immune system, which results in destruction of cells presenting such peptides. There has been significant advancement in recognizing antigens that are clinically significant (e.g., antigens associated with various types of cancer) and/or TCR sequences that bind the clinically significant antigens. However, in order to improve identification and selection of clinically significant peptides that will provoke a suitable response in a human T cell and/or of TCR capable of binding the clinically significant antigens (e.g., for adoptive immunotherapy of cancer, T cell vaccination for autoimmunity, etc.), there remains a need for in vivo and in vitro systems that mimic aspects of human immune system. Thus, there is a need for biological systems (e.g., genetically modified non-human animals and cells) that can display components of a human immune system, particularly components of the T cell immune response.

SUMMARY OF THE INVENTION

As disclosed herein, the thymus of genetically modified non-human animals comprising a substantially humanized T cell immune system has similar absolute numbers of thymocytes and CD3+ T cells as control animals. Additionally, these cells show comparable development into single positive T cells to control animals and are capable of generating a robust human cellular response against antigen, e.g., a viral antigen. The human cellular response of the non-human animals generally comprises activated non-human T cells expressing human or humanized T cell receptor (TCR) variable domains that recognize antigen presented in the peptide binding cleft formed by human leukocyte antigen (HLA) extracellular domains, which may be expressed on the surface of non-human antigen presenting cells. In some embodiments, the substantially humanized T cell immune system comprises

(A) a non-human T cell that expresses

-   -   (i) a T cell co-receptor polypeptide comprising a part or all of         the extracellular portion of a human T cell co-receptor, e.g., a         T cell co-receptor polypeptide comprising one or more human T         cell co-receptor extracellular domains such that the T cell         co-receptor polypeptide is capable of associating with and/or         associates with         -   (a) one or more extracellular domains of a human or             humanized HLA molecule (e.g., a first human HLA             extracellular domain that is a binding site for the T cell             co-receptor polypeptide and/or a second human HLA             extracellular domain that forms a peptide binding cleft,             e.g., with a third human HLA extracellular domain),         -   (b) an extracellular domain of a human or humanized TCR             variable domain (e.g., a human or humanized TCRα variable             domain and/or a human or humanized TCRβ variable domain that             is respectively encoded by at least one human TCRα and/or             TCRβ variable region gene segment), and/or         -   (c) an extracellular domain of a human TCR constant domain,             and     -   (ii) a T cell receptor (TCR) comprising at least a human TCR         variable domain; and optionally

(B) a non-human antigen presenting cell that presents antigen in the context of human HLA, e.g., a non-human antigen presenting cell that expresses on its cell surface at least one MHC molecule that comprises a peptide binding cleft formed by two human HLA extracellular domains, and is capable of activating and/or activates the non-human T cell.

In one aspect, the non-human T cell and the non-human antigen presenting cell are found in or isolated from the same non-human animal.

Accordingly, provided herein are non-human animals (e.g., rodents, e.g., mice or rats) genetically engineered to express

(A) a human or humanized T cell co-receptor (e.g., human or humanized CD4 and/or human or humanized CD8 (e.g., human or humanized CD8α and/or human or humanized CD8β)),

(B) a human or humanized major histocompatibility complex that associates with the human or humanized T cell co-receptor (e.g., human or humanized MHC II (e.g., human or humanized MHC II α and/or human or humanized MHC II β) that binds the human or humanized CD4 and/or human or humanized MHC I (e.g., human or humanized MHC Iα, and optionally human or humanized β2 microglobulin) that binds the human or humanized CD8), and/or

(C) a human or humanized T cell receptor (TCR);

as well as embryos, tissues, and cells expressing the same, and nucleic acids encoding the same. Also provided are methods of making and using the disclosed non-human animals.

In one aspect, provided is a genetically modified non-human animal, comprising

-   -   (A) a humanized CD4 co-receptor and/or a humanized CD8         co-receptor comprising a humanized CD8α polypeptide and a         humanized CD8β polypeptide (e.g., the non-human animal         comprises, e.g., in its germline genome, first nucleotide         sequence encoding a chimeric human/non-human CD4 polypeptide,         and/or a second nucleotide sequence encoding a chimeric         human/non-human CD8α polypeptide and a third nucleotide sequence         encoding a chimeric human/non-human CD8β polypeptide),         -   wherein each humanized T cell co-receptor polypeptide             comprises at least transmembrane and cytoplasmic domains of             a non-human T cell co-receptor, e.g., wherein the humanized             CD4 co-receptor comprises at least transmembrane and             cytoplasmic domains of a non-human CD4 co-receptor and/or             the humanized CD8 co-receptor comprises at least             transmembrane and cytoplasmic domains of non-human CD8α and             non-human CD8β polypeptides,         -   wherein each chimeric T cell co-receptor polypeptide             comprises part or all of an extracellular portion of a human             T cell co-receptor, e.g., one or more extracellular domains             of a human T cell co-receptor, e.g., at least an             extracellular domain of a human T cell co-receptor that             associates with an HLA molecule, e.g., wherein the humanized             CD4 co-receptor comprises the extracellular portion (or             parts thereof, e.g., extracellular domain(s)) of human CD4             that is responsible for interacting with MHC II, T cell             receptor variable domains, T cell receptor constant domains,             or a combination thereof, and/or e.g., wherein the humanized             CD8 co-receptor comprises the extracellular portions (or             parts thereof, e.g., extracellular domains) of human CD8α             and human CD8β that is responsible for interacting with MHC             I, T cell receptor variable domains, T cell receptor             constant domains, or a combination thereof;     -   (B) a human(ized) TCR (e.g., the non-human animal comprises,         e.g., in its germline genome, an unrearranged T cell receptor         (TCR) α variable gene locus comprising at least one human Vα         segment and at least one human Jα segment, operably linked to a         non-human TCRα constant gene sequence and/or an unrearranged         TCRβ variable gene locus comprising at least one human Vβ         segment, at least one human Dβ segment, and at least one human         Jβ segment, operably linked to a non-human TCRβ constant gene         sequence); and optionally,     -   (C) a human(ized) MHC II complex that associates with the         humanized CD4 co-receptor and/or a human(ized) MHC I complex         that associates with the humanized CD8 co-receptor (e.g., the         non-human animal comprises, e.g., in its germline genome, first         nucleic acid sequence encoding a chimeric human/non-human MHC         IIα polypeptide and a second nucleic acid sequence encoding a         chimeric human/non-human MHC II β polypeptide, and/or a third         nucleic acid sequence encoding a chimeric human/non-human MHC I         polypeptide),     -   wherein each chimeric MHC polypeptide comprises at least an         extracellular portion (or part thereof) of a human MHC         polypeptide (e.g., HLA polypeptide) that, either alone (e.g.,         MHC I) or when complexed with another chimeric MHC polypeptide         (e.g., MHC II α and MHC II β) is respectively capable of         associating with the human(ized) CD8 co-receptor or         human(ized)CD4 co-receptor and presenting peptide in the context         of HLA, e.g., wherein a humanized MHC II complex comprises (i) a         chimeric human/non-human MHC II α polypeptide comprising α1 and         α2 domains of a human HLA class II α polypeptide and the         transmembrane and cytoplasmic domains of a non-human HLA class         II α polypeptide and (ii) a chimeric human/non-human MHC II β         polypeptide comprises β1 and β2 domains of a human HLA class II         β polypeptide and the transmembrane and cytoplasmic domains of a         non-human HLA class II β polypeptide and/or wherein a humanized         MHC I complex comprises α1, α2, and α3 domains of a human MHC I         polypeptide, and optionally a human(ized) β2 microglobulin.

In some embodiments, the non-human animal comprises

-   -   (A) a humanized CD4 co-receptor and a humanized CD8 co-receptor         comprising a humanized CD8α polypeptide and a humanized CD8β         polypeptide (e.g., the non-human animal comprises, e.g., in its         germline genome, first nucleotide sequence encoding a chimeric         human/non-human CD4 polypeptide, a second nucleotide sequence         encoding a chimeric human/non-human CD8α polypeptide and a third         nucleotide sequence encoding a chimeric human/non-human CD8β         polypeptide),         -   wherein each humanized T cell co-receptor polypeptide             comprises at least transmembrane and cytoplasmic domains of             a non-human T cell co-receptor, e.g., wherein the humanized             CD4 co-receptor comprises at least transmembrane and             cytoplasmic domains of a non-human CD4 co-receptor and the             humanized CD8 co-receptor comprises at least transmembrane             and cytoplasmic domains of non-human CD8α and non-human CD8β             polypeptides,         -   wherein each chimeric T cell co-receptor polypeptide             comprises part or all of an extracellular portion of a human             T cell co-receptor, e.g., one or more extracellular domains             of a human T cell co-receptor, e.g., at least an             extracellular domain of a human T cell co-receptor that             associates with an HLA molecule, e.g., wherein the humanized             CD4 co-receptor comprises the extracellular portion (or             parts thereof, e.g., extracellular domain(s)) of human CD4             that is responsible for interacting with MHC II, T cell             receptor variable domains, T cell receptor constant domains,             or a combination thereof, and/or e.g., wherein the humanized             CD8 co-receptor comprises the extracellular portions (or             parts thereof, e.g., extracellular domains) of human CD8α             and human CD8β that are responsible for interacting with MHC             I, T cell receptor variable domains, T cell receptor             constant domains, or a combination thereof;     -   (B) a humanized TCR (e.g., the non-human animal comprises, e.g.,         in its germline genome, an unrearranged T cell receptor (TCR) α         variable gene locus comprising at least one human Vα segment and         at least one human Jα segment, operably linked to a non-human         TCRα constant gene sequence and/or an unrearranged TCRβ variable         gene locus comprising at least one human Vβ segment, at least         one human Dβ segment, and at least one human Jβ segment,         operably linked to a non-human TCRβ constant gene sequence); and     -   (C) a humanized MHC II complex that associates with the         humanized CD4 co-receptor and a humanized MHC I complex that         associates with the humanized CD8 co-receptor (e.g., the         non-human animal comprises, e.g., in its germline genome, first         nucleic acid sequence encoding a chimeric human/non-human MHC         IIα polypeptide, a second nucleic acid sequence encoding a         chimeric human/non-human MHC II β polypeptide and a third         nucleic acid sequence encoding a chimeric human/non-human MHC I         polypeptide),     -   wherein each chimeric MHC polypeptide comprises at least an         extracellular portion (or part thereof) of a human MHC         polypeptide (e.g., HLA polypeptide) that, either alone (e.g.,         MHC I) or when complexed with another chimeric MHC polypeptide         (e.g., MHC II α and MHC II β) is respectively capable of         associating with the humanized CD8 co-receptor or humanized CD4         co-receptor and presenting peptide in the context of HLA, e.g.,         wherein a humanized MHC II complex comprises (i) a chimeric         human/non-human MHC II α polypeptide comprising α1 and α2         domains of a human HLA class II α polypeptide and the         transmembrane and cytoplasmic domains of a non-human HLA class         II α polypeptide and (ii) a chimeric human/non-human MHC II β         polypeptide comprises β1 and β2 domains of a human HLA class II         β polypeptide the transmembrane and cytoplasmic domains of a         non-human HLA class II β polypeptide and (iii) a humanized MHC I         complex comprises α1, α2, and α3 domains of a human MHC I         polypeptide, and optionally a human(ized) β2 microglobulin         (e.g., the non-human animal further comprises a β2 microglobulin         locus encoding a polypeptide comprising a human β2 microglobulin         amino acid sequence, or a portion thereof).

In some embodiments, the first nucleotide sequence encoding a chimeric T cell CD4 co-receptor polypeptide is present at an endogenous CD4 T cell co-receptor locus, and/or the second nucleotide sequence encoding a chimeric T cell CD8α co-receptor polypeptide is present at an endogenous CD8α T cell co-receptor locus and the third nucleotide sequence encoding a chimeric T cell CD8β co-receptor polypeptide is present at an endogenous CD8β T cell co-receptor locus. Additional embodiments include a chimeric human/non-human CD4 polypeptide encoded by the gene set forth in FIG. 5A (e.g., wherein the human portion of the resulting chimeric human/non-human CD4 T cell co-receptor polypeptide comprises at least human Ig1, human Ig2 and human Ig3 domains, otherwise respectively referred to as D1, D2 and D3 domains) and/or a chimeric CD8 co-receptor encoded by the genes set forth in FIG. 5B (e.g., wherein the human portion of the chimeric CD8 co-receptor comprises all or substantially all of the extracellular portion of a human CD8 polypeptide (e.g., CD8α and/or CD8β), including human immunoglobulin V (IgV)-like a and β domains. In some embodiments, the human portion of the chimeric CD4 T cell co-receptor polypeptide comprises one or more extracellular domains of a human CD4 polypeptide (e.g., D1, D2, D3, D4, or any combination thereof) and the non-human portion of the chimeric CD4 T cell co-receptor polypeptide comprises the transmembrane and cytoplasmic domains of a non-human CD4 T cell co-receptor, the human portion of the chimeric CD8α polypeptide comprises an extracellular domain (e.g., an IgV-like domain) of a human CD8α polypeptide and the non-human portion of the chimeric CD8α polypeptide comprises the transmembrane and cytoplasmic domains of a non-human CD8α polypeptide, and/or the human portion of the CD8β polypeptide comprises an extracellular domain (e.g., an IgV-like domain) of the human CD8β polypeptide and the non-human portion of the chimeric CD8β T cell co-receptor polypeptide comprises the transmembrane and cytoplasmic domains of a non-human CD8β polypeptide.

In some embodiments, the first nucleic acid sequence encoding the human(ized) MHC II α is present at an endogenous non-human MHC II α locus and the second nucleic acid sequence encoding the human(ized) MHC II β is present at an endogenous non-human MHC II β locus, and/or the third nucleic acid sequence encoding the human(ized) MHC I is present at an endogenous non-human MHC I locus. In one aspect, the human(ized) MHC IIα polypeptide comprises the extracellular portion (or part thereof) of a human MHC IIα polypeptide (e.g., an HLA class IIα polypeptide), the human(ized) MHC II β polypeptide comprises the extracellular portion (or part thereof) of a human MHC II β polypeptide (e.g., an HLA class II β polypeptide) and/or the human(ized) MHC I polypeptide comprises the extracellular portion (or part thereof) of a human MHC I polypeptide (e.g., an HLA class I polypeptide). In some embodiments, the humanized MHC II α polypeptide comprises human MHC II α1 and α2 domains, the humanized MHC II β polypeptide comprises human MHC II β1 and β2 domains and/or the humanized MHC I polypeptide comprises human MHC I α1, α2, and α3 domains. In some embodiments, the first nucleic acid sequence encoding the chimeric human/non-human MHC II α polypeptide is operably linked to and/or expressed under regulatory control of endogenous non-human MHC II α promoter and regulatory elements, the second nucleic acid sequence encoding the chimeric human/non-human MHC II β polypeptide is e.g., operably linked to and/or expressed under regulatory control of endogenous non-human MHC II β promoter and regulatory elements, and/or the third nucleic acid sequence encoding the chimeric human/non-human MHC I polypeptide is operably linked to and/or expressed under regulatory control of an endogenous non-human MHC I promoter and regulatory elements. In additional embodiments, a non-human portion of the chimeric human/non-human MHC II α polypeptide comprises transmembrane and cytoplasmic domains of an endogenous non-human MHC II α polypeptide, a non-human portion of the chimeric human/non-human MHC II β polypeptide comprises transmembrane and cytoplasmic domains of an endogenous non-human MHC II β polypeptide and/or a non-human portion of the chimeric human/non-human MHC I polypeptide comprises transmembrane and cytoplasmic domains of an endogenous non-human MHC I polypeptide. Embodiments include non-human animals wherein the human portion of the proteins of chimeric human/non-human MHC II complex are derived from corresponding human HLA class II proteins selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP and/or wherein the human portion of the chimeric human/non-human MHC I polypeptide is derived from human HLA-A, human HLA-B, or human HLA-C. As non-limiting examples, in some embodiments, the chimeric MHC II α polypeptide comprises the extracellular portion, or a part thereof, of a HLA-DRα protein, a HLA-DQ α protein, or a HLA-DP α protein, the chimeric MHC II β polypeptide comprises the extracellular portion, or a part thereof, of a HLA-DRβ protein, a HLA-DQ β protein, or a HLA-DP β protein, and/or the chimeric MHC I polypeptide comprises the extracellular portion, or a part thereof, of a human HLA-A protein, a human HLA-B protein, or a human HLA-C protein. Non-human animals are also provided, wherein the human portions of the chimeric human/non-human MHC II proteins are derived from corresponding human HLA-DR proteins, e.g., the human portion of the human/non-human MHC II α polypeptide comprises α1 and α2 domains of the a chain of HLA-DR2 and the human portion of the human/non-human MHC II β polypeptide comprises β1 and β2 domains of the β chain of HLA-DR2 and/or wherein the human portion of the MHC I polypeptide is derived from a human HLA-A polypeptide, e.g., the human portion of the human/non-human MHC I polypeptide comprises the α1, α2, and α3 domains of a human HLA-A2 polypeptide, e.g., the α1, α2, and α3 domains of a human HLA-A2.1 polypeptide. Non-human animals wherein the non-human portions of the MHC II complex are derived from a murine H-2E encoding sequence and/or wherein the non-human portions of the MHC I polypeptide are derived from a murine H-2K encoding sequence are also provided. For example, the chimeric MHC II α polypeptide comprises the transmembrane and cytoplasmic domains of a murine H-2E α polypeptide, the chimeric MHC II β polypeptide comprises the transmembrane and cytoplasmic domains of a murine H-2E β polypeptide, and the chimeric MHC I polypeptide comprises the transmembrane and cytoplasmic domains of a murine H-2K polypeptide.

In some embodiments, the unrearranged TCRα variable gene locus is present at an endogenous TCRα variable gene locus and/or the unrearranged TCRβ variable gene locus is present at an endogenous TCRβ variable gene locus. In some embodiments, the unrearranged TCRα variable gene locus is present at an endogenous TCRα variable gene locus in the germline of the animal and/or the unrearranged TCRβ variable gene locus is present at an endogenous TCRβ variable gene locus in the germline of the animal. In some embodiments, the unrearranged TCR α variable region sequence comprises a mouse TCRA non-coding sequence and/or the unrearranged TCRβ variable region sequence comprises a mouse TCRB non-coding sequence. In some aspects, the unrearranged TCR α variable region sequence comprises at least one unrearranged human T cell variable region Vα segment and at least one unrearranged human T cell variable region Jα segment, e.g., operably linked to a mouse TCR α constant gene sequence and/or the unrearranged TCRβ variable region sequence comprises at least one unrearranged human T cell variable region Vβ segment, at least one unrearranged human T cell variable region Dβ segment, and at least one unrearranged human T cell variable region Jβ segment operably linked to a mouse TCRβ constant gene sequence, optionally at an endogenous mouse TCRβ variable gene locus. In some aspects, the at least one unrearranged human T cell variable region Vα segment, comprises a repertoire, e.g., a complete repertoire of human unrearranged Vα gene segments and the at least one unrearranged human T cell variable region Jα segment comprises a repertoire, e.g., a complete repertoire of human unrearranged Jα gene segments and/or at least one unrearranged human T cell variable region Vβ segment comprises a repertoire, e.g., a complete repertoire, of human unrearranged Vβ gene segments, at least one unrearranged human T cell variable region Dβ segment comprises a repertoire, e.g., a complete repertoire, of human unrearranged Dβ gene segments and at least one unrearranged human T cell variable region Jβ segment comprises a repertoire, e.g., a complete repertoire, of human unrearranged Jβ gene segments.

In some embodiments, the unrearranged TCRβ variable gene locus comprises a repertoire of human Vβ segments, an unrearranged human T cell variable region Dβ1 segment and an unrearranged human T cell variable region Dβ2 segment, and at least one unrearranged human T cell variable region Jβ1 segment and at least one unrearranged human T cell variable region Jβ2 segment, wherein the mouse TCRB non-coding sequence comprises a mouse TCRBD1-TCRBJ1 non-coding nucleic acid sequence between the at least one unrearranged human T cell variable region Dβ1 segment and the at least one unrearranged human T cell variable region Jβ1 segment and a mouse TCRBD2-TCRBJ2 non-coding nucleic acid sequence between the at least one unrearranged human T cell variable region Dβ2 segment and the at least one unrearranged human T cell variable region Jβ2 segment. In some embodiments, the unrearranged TCRβ variable gene locus comprises:

-   -   (a) a repertoire of human TCRBV segments, optionally wherein the         repertoire of human TRBV segments replace a corresponding         repertoire of endogenous TCRBV segments,     -   (b)(i) a humanized TCRBDJ1 cluster comprising an unrearranged         human TCRBD1 segment and (ii) any combination of an unrearranged         human TRBJ1-1 segment, an unrearranged human TRBJ1-2 segment, an         unrearranged human TCRBJ1-3 segment, an unrearranged human         TCRBJ1-4 segment, an unrearranged human TCRBJ1-5 segment, and an         unrearranged human TCRBJ1-6 segment,     -   wherein the humanized TCRBDJ1 cluster comprises a mouse TCRBDJ1         non-coding sequence between the unrearranged human TCRBD1         segment and any unrearranged human TCRBJ1 segment and a mouse         TCRBDJ1 non-coding sequence between any two consecutive         unrearranged human TCRBJ1 gene segments, optionally wherein the         unrearranged human TCRBD1 and TCRBJ1 gene segments flank the         same mouse TCRBDJ1 non-coding sequences as are normally flanked         by the corresponding mouse tcrbdj1 gene segments, and     -   (c)(i) a humanized TCRBDJ2 cluster comprising an unrearranged         human TCRBD2 segment and (ii) any combination of an unrearranged         human TRBJ2-1 segment, an unrearranged human TRBJ2-2 segment, an         unrearranged human TCRBJ2-3 segment, an unrearranged human         TCRBJ2-4 segment, an unrearranged human TCRBJ2-5 segment, an         unrearranged human TCRBJ2-6 segment, and an unrearranged human         TCRBJ2-7 segment,     -   wherein the humanized TCRBDJ2 cluster comprises a mouse TCRBDJ2         non-coding sequence between the unrearranged human TCRBD2         segment and any unrearranged human TCRBJ2 segment and a mouse         TCRBDJ2 non-coding sequence between any two consecutive         unrearranged human TCRBJ2 gene segments, optionally wherein the         unrearranged human TCRBD2 and TCRBJ2 gene segments flank the         same mouse TCRBDJ2 non-coding sequences as are normally flanked         by the corresponding mouse tcrbdj2 gene segments. In some         embodiments,     -   (I) the endogenous mouse TCRα variable gene locus comprises a         deletion selected from the group consisting of:         -   (a) a deletion of all endogenous TCR Vα gene segments,         -   (b) a deletion of all endogenous TCR Jα gene segments, and         -   (c) a combination thereof; or     -   (II) the endogenous mouse TCRβ variable gene locus comprises a         deletion selected from the group consisting of:         -   (a) a deletion of all contiguous endogenous TCR Vβ gene             segments (e.g., all endogenous TCR Vβ gene segments between             a 5′ trypsinogen cluster and a 3′ trypsinogen cluster), or a             deletion of all endogenous TCR Vβ gene segments,         -   (b) a deletion of all endogenous TCR Dβ gene segments,         -   (c) a deletion of all endogenous TCR Jβ gene segments, and         -   (d) a combination thereof; or     -   (III) the endogenous mouse TCRα variable gene locus comprises a         deletion selected from the group consisting of:         -   (a) a deletion of all endogenous TCR Vα gene segments,         -   (b) a deletion of all endogenous TCR Jα gene segments, and         -   (c) a combination thereof, and         -   the endogenous mouse TCRβ variable gene locus comprises a             deletion selected from the group consisting of:         -   (a) a deletion of all endogenous TCR Vβ gene segments,         -   (b) a deletion of all endogenous TCR Dβ gene segments,         -   (c) a deletion of all endogenous TCR Jβ gene segments, and         -   (d) a combination thereof.             In some embodiments,     -   (I) the endogenous mouse TCRα variable gene locus comprises a         replacement selected from the group consisting of:         -   (a) a replacement at least one endogenous T cell variable             region Vα gene segment with the at least one unrearranged             human T cell variable region Vα gene segment,         -   (b) a replacement of at least one endogenous T cell variable             region Jα gene segments with the at least unrearranged human             T cell variable region Jα segment, and         -   (c) a combination thereof; or     -   (II) the endogenous mouse TCRβ variable gene locus comprises a         replacement selected from the group consisting of:         -   (a) a replacement of at least one endogenous T cell variable             region Vβ gene segment with the at least one unrearranged             human T cell variable region Vβ segment,         -   (b) a replacement of at least one endogenous T cell variable             region Dβ gene segment with the at least one unrearranged             human T cell variable region Dβ segment,         -   (c) a replacement of at least one endogenous T cell variable             region Jβ gene segment with the at least one unrearranged             human T cell variable region Jβ segment, and         -   (d) a combination thereof; or     -   (III) the endogenous mouse TCRα variable gene locus comprises a         replacement selected from the group consisting of:         -   (a) a replacement at least one endogenous T cell variable             region Vα gene segment with the at least one unrearranged             human T cell variable region Vα gene segment,         -   (b) a replacement of at least one endogenous T cell variable             region Jα gene segments with the at least unrearranged human             T cell variable region Jα segment, and         -   (c) a combination thereof, and         -   the endogenous mouse TCRβ variable gene locus comprises a             replacement selected from the group consisting of:         -   (a) a replacement of at least one endogenous T cell variable             region Vβ gene segment with the at least one unrearranged             human T cell variable region Vβ segment,         -   (b) a replacement of at least one endogenous T cell variable             region Dβ gene segment with the at least one unrearranged             human T cell variable region Dβ segment,         -   (c) a replacement of at least one endogenous T cell variable             region Jβ gene segment with the at least one unrearranged             human T cell variable region Jβ segment, and         -   (d) a combination thereof.             In some embodiments,     -   (I) the endogenous mouse TCRα variable gene locus comprises:         -   (a) a replacement of all endogenous T cell variable region             Vα gene segment with the at least one unrearranged human T             cell variable region Vα gene segment, optionally wherein the             at least one unrearranged human T cell variable region Vα             gene segment comprises a plurality or all unrearranged human             T cell variable region gene segments from TRAV1-1 to TRAV41,         -   (b) a replacement of all endogenous T cell variable region             Jα gene segments with the at least one unrearranged human T             cell variable region Jα segment, optionally wherein the at             least one unrearranged human T cell variable region Jα             segment comprises a plurality or all unrearranged human T             cell variable region gene segments from TRAJ1 to TRAJ61, or         -   (c) a combination thereof;     -   (II) the endogenous mouse TCRβ variable gene locus comprises:         -   (a) a replacement of all contiguous endogenous T cell             variable region VR gene segments with the at least one             unrearranged human T cell variable region Vβ segment,             optionally wherein the at least one unrearranged human T             cell variable region Vβ gene segment comprises a plurality             or all unrearranged human T cell variable region gene             segments from TRBV1 to TRBV29-1,         -   (b) a replacement of all endogenous T cell variable region             Dβ gene segments with the at least one unrearranged human T             cell variable region Dβ gene segment, optionally wherein the             at least one unrearranged human T cell variable region Dβ             gene segment comprises an unrearranged human T cell variable             region Dβ1 gene segment and/or an unrearranged human T cell             variable region Dβ2 gene segment,         -   (c) a replacement of all endogenous T cell variable region             Jβ gene segments with the at least one unrearranged human T             cell variable region Jβ segment, optionally wherein the at             least one unrearranged human T cell variable region Jβ             segment comprises a plurality or all unrearranged human Jβ             segment from TRBJ1-1 to TRBJ1-6 and/or a plurality or all             unrearranged human Jβ segments from TRBJ2-1 to TRBJ2-7, or         -   (d) a combination thereof; or     -   (III) the endogenous mouse TCRα variable gene locus comprises:         -   (a) a replacement of all endogenous T cell variable region             Vα gene segments with the at least one unrearranged human T             cell variable region Vα gene segment, optionally wherein the             at least one unrearranged human T cell variable region Vα             gene segment comprises a plurality or all unrearranged human             T cell variable region gene segments from TRAV1-1 to TRAV41,         -   (b) a replacement of all endogenous T cell variable region             Jα gene segments with the at least unrearranged human T cell             variable region Jα segment, optionally wherein the at least             one unrearranged human T cell variable region Jα segment             comprises a plurality or all unrearranged human T cell             variable region gene segments from TRAJ1 to TRAJ61, or         -   (c) a combination thereof, and     -   the endogenous mouse TCRβ variable gene locus comprises:         -   (a) a replacement of all contiguous endogenous T cell             variable region Vβ gene segments with the at least one             unrearranged human T cell variable region Vβ segment,             optionally wherein the at least one unrearranged human T             cell variable region Vβ gene segment comprises a plurality             or all unrearranged human T cell variable region gene             segments from TRBV1 to TRBV29-1,         -   (b) a replacement of all endogenous T cell variable region             Dβ gene segments with the at least one unrearranged human T             cell variable region Dβ gene segment, optionally wherein the             at least one unrearranged human T cell variable region Dβ             gene segment comprises an unrearranged human T cell variable             region Dβ1 gene segment and/or an unrearranged human T cell             variable region Dβ2 gene segment,         -   (c) a replacement of all endogenous T cell variable region             Jβ gene segments with the at least one unrearranged human T             cell variable region Jβ segment, optionally wherein the at             least one unrearranged human T cell variable region Jβ             segment comprises a plurality or all unrearranged human Jβ             segments from TRBJ1-1 to TRBJ1-6 and/or a plurality or all             unrearranged human Jβ segments from TRBJ2-1 to TRBJ2-7, or         -   (d) a combination thereof.             In some embodiments,     -   (I) the endogenous mouse TCRα variable gene locus comprises:         -   (a) a replacement of all endogenous T cell variable region             Vα gene segments with all unrearranged human T cell variable             region gene segments from TRAV1-1 to TRAV41,         -   (b) a replacement of all endogenous T cell variable region             Jα gene segments with all unrearranged human T cell variable             region gene segments from TRAJ1 to TRAJ61, or         -   (c) a combination thereof;     -   (II) the endogenous mouse TCRβ variable gene locus comprises:         -   (a) a replacement of all contiguous endogenous T cell             variable region Vβ gene segments with all unrearranged human             T cell variable region gene segments from TRBV1 to TRBV29-1,         -   (b) a replacement of endogenous T cell variable region Dβ1             gene segment with unrearranged human T cell variable region             Dβ1 gene segment and a replacement of endogenous T cell             variable region Dβ2 gene segment with unrearranged human T             cell variable region Dβ2 gene segment,         -   (c) a replacement of:             -   an endogenous TRBJ1-1 gene segment with an unrearranged                 human TRBJ1-1 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBD1 segment and a mouse                 TRBJ1-1 segment,             -   an endogenous TRBJ1-2 gene segment with an unrearranged                 human TRBJ1-2 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ1-1 segment and a                 mouse TRBJ1-2 segment,             -   an endogenous TRBJ1-3 gene segment with an unrearranged                 human TRBJ1-3 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ1-2 segment and a                 mouse TRBJ1-3 segment,             -   an endogenous TRBJ1-4 gene segment with an unrearranged                 human TRBJ1-4 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ1-3 segment and a                 mouse TRBJ1-4 segment,             -   an endogenous TRBJ1-5 gene segment with an unrearranged                 human TRBJ1-5 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ1-4 segment and a                 mouse TRBJ1-5 segment,             -   an endogenous TRBJ1-6 gene segment with an unrearranged                 human TRBJ1-6 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ1-5 segment and a                 mouse TRBJ1-6 segment,             -   an endogenous TRBJ2-1 gene segment with an unrearranged                 human TRBJ2-1 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBD2 segment and a mouse                 TRB2-1 segment,             -   an endogenous TRBJ2-2 gene segment with an unrearranged                 human TRBJ2-2 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ2-1 segment and a                 mouse TRBJ2-2 segment,             -   an endogenous TRBJ2-3 gene segment with an unrearranged                 human TRBJ2-3 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ2-2 segment and a                 mouse TRBJ2-3 segment,             -   an endogenous TRBJ2-4 gene segment with an unrearranged                 human TRBJ2-4 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ2-3 segment and a                 mouse TRBJ2-4 segment,             -   an endogenous TRBJ2-5 gene segment with an unrearranged                 human TRBJ2-5 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ2-4 segment and a                 mouse TRBJ2-5 segment,             -   an endogenous TRBJ2-6 gene segment with an unrearranged                 human TRBJ2-6 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ2-5 segment and a                 mouse TRBJ2-6 segment, and             -   an endogenous TRBJ2-7 gene segment with an unrearranged                 human TRBJ2-7 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ2-6 segment and a                 mouse TRBJ2-7 segment, or         -   (d) a combination thereof; or     -   (III) the endogenous mouse TCRα variable gene locus comprises:         -   (a) a replacement of all endogenous T cell variable region             Vα gene segment with all unrearranged human T cell variable             region gene segments from TRAV1-1 to TRAV41,         -   (b) a replacement of all endogenous T cell variable region             Jα gene segments with all unrearranged human T cell variable             region gene segments from TRAJ1 to TRAJ61, or         -   (c) a combination thereof, and     -   the endogenous mouse TCRβ variable gene locus comprises:         -   (a) a replacement of all contiguous endogenous T cell             variable region Vβ gene segments with all unrearranged human             T cell variable region gene segments from TRBV1 to TRBV29-1,         -   (b) a replacement of endogenous T cell variable region Dβ1             gene segment with unrearranged human T cell variable region             Dβ1 gene segment and a replacement of endogenous T cell             variable region Dβ2 gene segment with unrearranged human T             cell variable region Dβ2 gene segment,         -   (c) a replacement of:             -   an endogenous TRBJ1-1 gene segment with an unrearranged                 human TRBJ1-1 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBD1 segment and a mouse                 TRBJ1-1 segment,             -   an endogenous TRBJ1-2 gene segment with an unrearranged                 human TRBJ1-2 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ1-1 segment and a                 mouse TRBJ1-2 segment,             -   an endogenous TRBJ1-3 gene segment with an unrearranged                 human TRBJ1-3 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ1-2 segment and a                 mouse TRBJ1-3 segment,             -   an endogenous TRBJ1-4 gene segment with an unrearranged                 human TRBJ1-4 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ1-3 segment and a                 mouse TRBJ1-4 segment,             -   an endogenous TRBJ1-5 gene segment with an unrearranged                 human TRBJ1-5 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ1-4 segment and a                 mouse TRBJ1-5 segment,             -   an endogenous TRBJ1-6 gene segment with an unrearranged                 human TRBJ1-6 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ1-5 segment and a                 mouse TRBJ1-6 segment,             -   an endogenous TRBJ2-1 gene segment with an unrearranged                 human TRBJ2-1 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBD2 segment and a mouse                 TRBJ2-1 segment,             -   an endogenous TRBJ2-2 gene segment with an unrearranged                 human TRBJ2-2 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ2-1 segment and a                 mouse TRBJ2-2 segment,             -   an endogenous TRBJ2-3 gene segment with an unrearranged                 human TRBJ2-3 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ2-2 segment and a                 mouse TRBJ2-3 segment,             -   an endogenous TRBJ2-4 gene segment with an unrearranged                 human TRBJ2-4 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ2-3 segment and a                 mouse TRBJ2-4 segment,             -   an endogenous TRBJ2-5 gene segment with an unrearranged                 human TRBJ2-5 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ2-4 segment and a                 mouse TRBJ2-5 segment,             -   an endogenous TRBJ2-6 gene segment with an unrearranged                 human TRBJ2-6 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ2-5 segment and a                 mouse TRBJ2-6 segment, and             -   an endogenous TRBJ2-7 gene segment with an unrearranged                 human TRBJ2-7 gene segment, optionally wherein the mouse                 TCRB non-coding sequence comprises a mouse non-coding                 sequence found between a mouse TRBJ2-6 segment and a                 mouse TRBJ2-7 segment; or         -   (d) a combination thereof.             In some embodiments,     -   (I) the endogenous mouse TCRα variable gene locus comprises:         -   (a) a replacement of all endogenous T cell variable region             Vα gene segments with all unrearranged human T cell variable             region gene segments from TCRAV1-1 to TCRAV41, and         -   (b) a replacement of all endogenous T cell variable region             Jα gene segments with all unrearranged human T cell variable             region gene segments from TCRAJ1 to TCRAJ61, or (c) a             combination thereof; and     -   (II) the endogenous mouse TCRβ variable gene locus comprises:         -   (a) a replacement of all contiguous endogenous T cell             variable region Vβ gene segments, e.g., all contiguous             endogenous T cell variable region Vβ gene segments between a             first 5′ trypsinogen cluster and a second 3′ trypsinogen             cluster, with all unrearranged human T cell variable region             gene segments from TRBV1 to TRBV29-1,         -   (b)(i) a replacement of an endogenous tcrbdj1 cluster with a             humanized TCRBDJ1 cluster comprising an unrearranged human             TCRBD1 segment and (ii) each of an unrearranged human             TCRBJ1-1 segment, an unrearranged human TCRBJ1-2 segment, an             unrearranged human TCRBJ1-3 segment, an unrearranged human             TCRBJ1-4 segment, an unrearranged human TCRBJ1-5 segment,             and an unrearranged human TCRBJ1-6 segment,     -   wherein the humanized TCRBDJ1 cluster comprises a mouse TCRBDJ1         non-coding sequence between the unrearranged human TCRBD1         segment and the unrearranged human TCRBJ1-1 segment and a mouse         TCRBDJ1 non-coding sequence between any two consecutive         unrearranged human TCRBJ1 gene segments, optionally wherein the         unrearranged human TCRBD1 and TCRBJ1 gene segments flank the         same mouse TCRBDJ1 non-coding sequences as are normally flanked         by the corresponding mouse tcrbdj1 gene segments, and         -   (c)(i) a replacement of an endogenous tcrbdj2 cluster a             humanized TCRBDJ2 cluster comprising an unrearranged human             TCRBD2 segment and (ii) each of an unrearranged human             TRBJ2-1 segment, an unrearranged human TRBJ2-2 segment, an             unrearranged human TCRBJ2-3 segment, an unrearranged human             TCRBJ2-4 segment, an unrearranged human TCRBJ2-5 segment, an             unrearranged human TCRBJ2-6 segment, and an unrearranged             human TCRBJ2-7 segment,     -   wherein the humanized TCRBDJ2 cluster comprises a mouse TCRBDJ2         non-coding sequence between the unrearranged human TCRBD2         segment and any unrearranged human TCRBJ2 segment and a mouse         TCRBDJ2 non-coding sequence between any two consecutive         unrearranged human TCRBJ2 gene segments, optionally wherein the         unrearranged human TCRBD2 and TCRBJ2 gene segments flank the         same mouse TCRBDJ2 non-coding sequences as are normally flanked         by the corresponding mouse tcrbdj2 gene segments.

In some embodiments (e.g., where an endogenous TCRβ variable gene locus, e.g., an endogenous TCRβ mouse variable gene locus, comprises a replacement of one or all of the contiguous endogenous T cell variable region Vβ gene segments, e.g., one or all contiguous endogenous T cell variable region Vβ gene segments between a first 5′ trypsinogen cluster and a second 3′ trypsinogen cluster, with one or all unrearranged human T cell variable region gene segments from TRBV1 to TRBV29-1), an endogenous TCRβ variable gene locus may comprise a replacement of one or more non-contiguous endogenous Vβ gene segments (e.g., an endogenous mouse TCRBV31 gene segment) with a human TCRBV gene segment (e.g., a replacement of a mouse TCRBV31 gene segment with an orthologous human TCRBV30 gene segment).

In some embodiments, the human unrearranged Vα and Jα gene segments rearrange to form a rearranged human Vα/Jα sequence and/or the human unrearranged Vβ, Dβ and Jβ gene segment rearrange to form a rearranged human Vβ/Dβ/Jβ sequence, optionally wherein the TCRβ chain is encoded by a rearranged Vβ/Dβ2/Jβ2 sequence (e.g., a rearranged Vβ/Dβ2/Jβ2 sequence derived from a TCRBJD2 cluster). In some embodiments, a non-human animal as disclosed herein expresses a T cell receptor comprising a human TCRα variable region and/or a human TCRβ variable region on the surface of a T cell. In some embodiments, endogenous non-human Vα and Jα segments are incapable of rearranging to form a rearranged Vα/Jα sequence and/or endogenous non-human Vβ, Dβ, and Jβ segments are incapable of rearranging to form a rearranged Vβ/DβJβ sequence, e.g., the animal may lack a functional endogenous non-human TCRα variable locus and/or the animal may lack a functional endogenous non-human TCRβ variable locus, e.g., the animal comprises (a) a deletion of all or substantially all functional endogenous Vα gene segments, (b) a deletion of all or substantially all functional endogenous Jα gene segments, (c) a deletion of all or substantially all functional endogenous Vβ gene segments, (d) a deletion of all or substantially all functional endogenous Dβ gene segments, (e) a deletion of all or substantially all functional endogenous Jβ gene segments, and/or (f) a combination thereof. In some embodiments, the endogenous non-human TCRα variable locus lacks all or substantially all functional endogenous Vα gene segments and/or lacks all or substantially all functional endogenous Jα gene segments; and/or the endogenous non-human TCRβ variable locus (a) lacks all or substantially all functional endogenous Vβ gene segments, (b) lacks all or substantially all functional endogenous Dβ gene segments, (c) lacks all or substantially all functional endogenous Jβ gene segments, or (d) any combination of (a), (b), and (c).

In some embodiments, wherein at least 10% of the TCR expressed by the mouse is derived from gene segments from the TCRBDJ1 cluster and at least 10% of the TCR expressed by the mouse is derived from gene segments from the TCRBDJ2 cluster.

In some embodiments, the first, second and/or third nucleotide sequence(s) respectively encoding the chimeric T cell CD4, CD8α and/or CD8 β co-receptor polypeptide(s) is present at endogenous T cell co-receptor loci, e.g., endogenous CD4, CD8α and/or CD8 β co-receptor loci respectively; the unrearranged TCRα variable gene locus is present at an endogenous TCRα variable gene locus; the unrearranged TCRβ variable gene locus is present at an endogenous TCRβ variable gene locus; and/or the first, second and/or third nucleic acid sequence(s) respectively encoding the chimeric MHC II α, MHC II β, and/or MHC I polypeptide(s) is present at endogenous MHC loci; e.g., MHC II α, MHC II β, and/or MHC I loci, respectively. In some embodiments, the nucleotide sequence(s) encoding the chimeric T cell co-receptor(s), the unrearranged TCRα variable gene locus, the unrearranged TCRβ variable gene locus and/or the nucleic acid sequence(s) encoding the chimeric MHC molecule(s) may be operably linked to non-human promoter and/or regulatory sequences. For example, the first nucleotide sequence may be operably linked to and/or expressed under regulatory control of endogenous non-human CD4 promoter and regulatory elements, the second nucleotide sequence may be operably linked to and/or expressed under regulatory control of endogenous non-human CD8α promoter and regulatory elements, and and/or the third nucleotide sequence may operably linked to and/or expressed under regulatory control of endogenous non-human CD8β promoter and regulatory elements; the unrearranged TCRα variable gene locus may be operably linked to and/or expressed under regulatory control of endogenous TCRα regulatory and/or promoter elements and the unrearranged TCRβ variable gene locus may be operably linked to and/or expressed under regulatory control of endogenous TCRβ regulatory and/or promoter elements; the first nucleic acid sequence may be operably linked to and/or expressed under regulatory control of endogenous non-human MHC II α promoter and regulatory elements, the second nucleic acid sequence may be operably linked to and/or expressed under regulatory control of endogenous non-human MHC II β promoter and regulatory elements, and the third nucleic acid sequence may operably linked to and/or expressed under regulatory control of an endogenous non-human MHC I promoter and regulatory elements.

In some embodiments, a nucleotide sequence encoding the extracellular portion (or parts thereof, e.g., D1, D2, D3 and/or D4) of the human CD4 polypeptide replaces a sequence encoding the extracellular portion (or parts thereof, e.g., D1, D2, D3 and/or D4) of an endogenous non-human (mouse) CD4 co-receptor polypeptide, and may be operably linked to endogenous non-human (mouse) CD4 transmembrane and cytoplasmic domain encoding sequences, at the endogenous non-human (mouse) CD4 co-receptor locus; a nucleotide sequence encoding all or part of the extracellular portion of a human CD8α polypeptide replaces a sequence encoding all or part of an extracellular portion of an endogenous non-human (mouse) T cell CD8α polypeptide, and may be operably linked to endogenous non-human (mouse) CD8α transmembrane and cytoplasmic domain encoding sequences, at the endogenous non-human (mouse) CD8α locus; a nucleotide sequence encoding all or part of the extracellular domain of a human CD8β polypeptide replaces a sequence encoding all or part of an extracellular domain of an endogenous non-human (mouse) T cell CD8β polypeptide and may be operably linked to endogenous non-human CD8β transmembrane and cytoplasmic domain encoding sequences, at the endogenous CD8β locus; an unrearranged TCRα variable gene locus replaces one or more endogenous Vα and/or Jα gene segments at an endogenous non-human (mouse) TCRα variable gene locus; an unrearranged TCRβ variable gene locus replaces one or more endogenous Vβ, Dβ and/or Jα gene segments at an endogenous non-human (mouse) TCRβ variable gene locus; a nucleic acid sequence encoding the extracellular portion (or parts thereof, e.g., α1 and α2 domains) of a human MHC II α polypeptide replaces a sequence encoding the extracellular portion (or parts thereof, e.g., α1 and α2 domains) of an endogenous non-human (mouse) MHC II α polypeptide, and may be operably linked to endogenous non-human (mouse) MHC II α transmembrane and cytoplasmic domain encoding sequences, at an endogenous non-human (mouse) MHC II α locus; a nucleic acid sequence encoding the extracellular portion (or parts thereof, e.g., β1 and β2 domains) of a human MHC II β polypeptide replaces a sequence encoding the extracellular portion (or parts thereof, e.g., β1 and β2 domains) of an endogenous non-human (mouse) MHC II β polypeptide, and may be operably linked to endogenous non-human (mouse) MHC II β transmembrane and cytoplasmic domain encoding sequences, at an endogenous non-human (mouse) MHC II β locus; and/or a nucleic acid sequence encoding the extracellular portion (or parts thereof, e.g., α1, α2 and/or α3 domains) of a human MHC I polypeptide replaces a sequence encoding the extracellular portion (or parts thereof, e.g., α1, α2 and/or α3 domains) of an endogenous non-human (mouse) MHC I polypeptide, and may be operably linked to endogenous non-human (mouse) MHC I transmembrane and cytoplasmic domain encoding sequences, at an endogenous non-human (mouse) MHC I locus.

In some embodiments, a genetically modified non-human animal as disclosed herein does not express a functional endogenous non-human T cell CD4 co-receptor from its endogenous locus, does not express a functional endogenous non-human T cell CD8 co-receptor from its endogenous CD8 locus, does not express a functional TCRα variable domain from an endogenous TCRα variable locus, does not express a function TCRβ variable domain from an endogenous TCRβ variable locus, does not express an extracellular domain of an endogenous MHC II complex from an endogenous MHC II locus (e.g., on a cell surface) and/or does not express an extracellular domain of an endogenous MHC I polypeptide from an endogenous MHC I locus (e.g., on a cell surface).

Any non-human animal disclosed herein may further comprise a β2 microglobulin locus encoding a polypeptide comprising a human or humanized β2 microglobulin amino acid sequence, wherein the non-human animal expresses the human or humanized β2 microglobulin polypeptide. In some embodiments, the non-human animal does not express a functional endogenous non-human animal β2 microglobulin polypeptide from an endogenous non-human β2 microglobulin locus. In some embodiments, the β2 microglobulin locus is operably linked to endogenous non-human β2 microglobulin regulatory elements. In one embodiment, the β2 microglobulin locus comprises a nucleotide sequence set forth in exon 2, exon 3, and exon 4 (e.g., exon 2 to exon 4) of a human β2 microglobulin gene, and optionally, the β2 microglobulin locus further comprises a nucleotide sequence set forth in exon 1 of a non-human, e.g., rodent, β2 microglobulin gene.

Non-human animals as provided herein may be a rodent, e.g., a mouse or a rat.

Also provided herein is a mouse that expresses chimeric human/murine T cell CD4, CD8α, and CD8β co-receptor polypeptides each respectively comprising murine CD4, CD8α, and CD8β transmembrane and cytoplasmic domains; a T cell receptor comprising a human TCRα variable region and a human TCRβ variable region on the surface of a T cell; chimeric human/murine MHC IIα, MHC II β, and MHC I polypeptides each respectively comprising extracellular domains of a human MHC II α (e.g., human HLA class II α1 and α2 domains), MHC II β (human HLA class II β1 and β2 domains), and MHC I polypeptide (e.g., human HLA class I α1, α2, and α3 domains); and optionally a human or humanized β2 microglobulin polypeptide. In one embodiment, provided herein are non-human animals, e.g., mice, wherein the first nucleic acid sequence encodes an a chain of a chimeric human/murine HLA-DR/H-2E polypeptide, the second nucleotide sequence encodes a β chain of a chimeric HLA-DR/H-2E polypeptide, and the third nucleic acid sequence encodes a chimeric human/murine HLA-A/H-2K polypeptide, and wherein the mouse expresses HLA-A/H-2K and HLA-DR/H-2E proteins. In some embodiments, at least 10% of the TCR expressed by the mouse is derived from gene segments from the TCRBDJ1 cluster and at least 10% of the TCR expressed by the mouse is derived from gene segments from the TCRBDJ2 cluster.

Also provided herein is a non-human animal comprising a substantially humanized T cell immune system, e.g., wherein the substantially humanized T cell immune system mounts a substantially humanized T cell immune response against an antigen. In some embodiments, the substantially humanized T cell immune response comprises activated T cells expressing human T cell receptor (TCR) variable domains that recognize antigen presented in the context of human leukocyte antigen (HLA) extracellular domains and/or antigen presenting cells that present antigen in the context of HLA extracellular domains. In some embodiments, the substantially humanized T cell immune system comprises: (a) a non-human T cell that expresses a T cell co-receptor polypeptide comprising a human T cell co receptor domain that binds to a human HLA molecule and/or a T cell receptor (TCR) comprising a TCR variable domain that is encoded by at least one human TCR variable region gene segment; and (b) a non-human antigen presenting cell that presents antigen in the context of human HLA and activates the non-human T cell.

Also provided are methods of making and using the non-human animals disclosed herein. Generally, methods of making a genetically modified non-human animal as disclosed herein comprise (a) introducing into the genome of the non-human animal a first nucleotide sequence encoding a chimeric human/non-human T cell co-receptor polypeptide (e.g., a chimeric CD4 polypeptide), and/or a second nucleotide sequence encoding a second chimeric human/non-human T cell co-receptor polypeptide (e.g., a chimeric CD8α polypeptide) and a third nucleotide sequence encoding a third chimeric human/non-human T cell co-receptor polypeptide (e.g., a CD8β polypeptide), wherein a non-human portion of each chimeric T cell co-receptor polypeptide comprises at least transmembrane and cytoplasmic domains of a non-human T cell co-receptor, and wherein a human portion of each chimeric polypeptide comprises an extracellular portion (or part thereof, e.g., one or more domains) of a human T cell co-receptor; (b) inserting into the genome of the non-human animal an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCRα constant gene sequence, optionally wherein the unrearranged TCR α variable region comprises a mouse TCRA non-coding sequence, and/or an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCRβ constant gene sequence, optionally wherein the unrearranged TCR α variable region comprises a mouse TCRA non-coding sequence; and optionally (c) placing into the genome a first nucleic acid sequence encoding a first chimeric human/non-human MHC polypeptide (e.g., a chimeric MHC IIα polypeptide), a second nucleic acid sequence encoding a second chimeric human/non-human MHC polypeptide (e.g., a chimeric MHC II β polypeptide) and/or a third nucleic acid sequence encoding a third chimeric human/non-human MHC polypeptide (e.g., a chimeric MHC I polypeptide) and/or (d) adding into the genome of the non-human animal a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide. In some embodiments, the first nucleotide sequence encodes the extracellular portion, or a part thereof, of human CD4 operably linked to at least transmembrane and cytoplasmic domains of a non-human CD4 co-receptor, the second nucleotide sequence encodes the extracellular portion, or a part thereof, of human CD8α and at least the transmembrane and cytoplasmic domains of a non-human CD8α, the third nucleotide sequence encodes the extracellular portion, or a part thereof, of human CD8β and at least the transmembrane and cytoplasmic domains of non-human CD8β, the first nucleic acid sequence encodes the extracellular portion (or part thereof) of a human HLA class II α polypeptide and at least the transmembrane and cytoplasmic domains of a non-human MHC II α polypeptide, the second nucleic acid sequence encodes the extracellular portion (or part thereof) of a human HLA class II β polypeptide and at least the transmembrane and cytoplasmic domains of a non-human MHC II β polypeptide, the third nucleic acid sequence encodes the extracellular portion (or part thereof) of a human HLA class I polypeptide and the transmembrane and cytoplasmic domains of a non-human MHC I polypeptide, and the β2 microglobulin locus comprises a nucleotide sequence set forth in exons 2 to 4 of the human β2 microglobulin gene, e.g., nucleotide sequences set forth in exons 2, 3, and 4 of the human β2 microglobulin gene.

Some methods of making non-human animals include embodiments that comprise replacing a contiguous mouse TCRB sequence comprising a mouse TCRBD gene segment and a mouse TCRBJ gene segment with a nucleic acid sequence comprising the at least one unrearranged human T cell variable region Dβ segment, a mouse TCRBD-TCRBJ non-coding nucleic acid sequence, and the at least one unrearranged human T cell variable region Jβ segment, such that the at least one unrearranged human T cell variable region DR segment, the mouse TCRBD-TCRBJ non-coding nucleic acid sequence, and the at least one unrearranged human T cell variable region Jβ segment are operably linked to the mouse TCRβ constant gene sequence. In some embodiments, the contiguous mouse TCRB sequence comprises

(a) a mouse TCRBD1 gene segment and a mouse TCRBJ1-6 gene segment and/or

(b) a mouse TCRBD2 gene segment and a mouse TCRBJ2-7 gene segment, and wherein the nucleic acid comprises:

(c) a humanized TCRBDJ1 cluster comprising an unrearranged human TCRBD1 gene segment, an unrearranged human TCRBJ1 gene segment, and a mouse TCRBDJ1 non-coding sequence, wherein the humanized TCRBDJ1 cluster comprises:

-   -   an unrearranged human TRBJ1-1 gene segment and a mouse TCRB         non-coding sequence between the unrearranged human TRBD1 gene         segment and the unrearranged human TRBJ1-1 gene segment,     -   an unrearranged human TRBJ1-1 gene segment, an unrearranged         human TRBJ1-2 gene segment, and a mouse TCRB non-coding sequence         between the unrearranged human TRBJ1-1 gene segment and the         unrearranged human TRBJ1-2 gene segment,     -   an unrearranged human TRBJ1-2 gene segment, an unrearranged         human TRBJ1-3 gene segment, and a mouse TCRB non-coding sequence         between the unrearranged human TRBJ1-2 gene segment and the         unrearranged human TRBJ1-3 gene segment,     -   an unrearranged human TRBJ1-3 gene segment, an unrearranged         human TRBJ1-4 gene segment, and a mouse TCRB non-coding sequence         between the unrearranged human TRBJ1-3 gene segment and the         unrearranged human TRBJ1-4 gene segment,     -   an unrearranged human TRBJ1-4 gene segment, an unrearranged         human TRBJ1-5 gene segment, and a mouse TCRB non-coding sequence         between the unrearranged human TRBJ1-4 gene segment and the         unrearranged human TRBJ1-5 gene segment,     -   an unrearranged human TRBJ1-5 gene segment, an unrearranged         human TRBJ1-6 gene segment, and a mouse TCRB non-coding sequence         between the unrearranged human TRBJ1-5 gene segment and the         unrearranged human TRBJ1-6 gene segment, or     -   any combination thereof (e.g., wherein the humanized TCRBDJ1         cluster comprises (i) an unrearranged human TCRBD1 segment         and (ii) any combination of an unrearranged human TRBJ1-1         segment, an unrearranged human TRBJ1-2 segment, an unrearranged         human TCRBJ1-3 segment, an unrearranged human TCRBJ1-4 segment,         an unrearranged human TCRBJ1-5 segment, and an unrearranged         human TCRBJ1-6 segment; and wherein the humanized TCRBDJ1         cluster comprises a mouse TCRBDJ1 non-coding sequence between         the unrearranged human TCRBD1 segment and any unrearranged human         TCRBJ1 segment and a mouse TCRBDJ1 non-coding sequence between         any two consecutive unrearranged human TCRBJ1 gene segments,         optionally wherein the unrearranged human TCRBD1 and TCRBJ1 gene         segments flank the same mouse TCRBDJ1 non-coding sequences as         are normally flanked by the corresponding mouse tcrbdj1 gene         segments); and/or

(d) a humanized TCRBDJ2 cluster comprising an unrearranged human TCRBD2 gene segment, an unrearranged human TCRBJ2 gene segment, and a mouse TCRBDJ2 non-coding sequence, wherein the humanized TCRBDJ2 cluster comprises:

-   -   an unrearranged human TRBJ2-1 gene segment and a mouse TCRB         non-coding sequence between the unrearranged human TRBD2 gene         segment and the unrearranged human TRBJ2-1 gene segment,     -   an unrearranged human TRBJ2-1 gene segment, an unrearranged         human TRBJ2-2 gene segment, and a mouse TCRB non-coding sequence         between the unrearranged human TRBJ2-1 gene segment and the         unrearranged human TRBJ2-2 gene segment,     -   unrearranged human TRBJ2-2 gene segment, an unrearranged human         TRBJ2-3 gene segment, and a mouse TCRB non-coding sequence         between the unrearranged human TRBJ2-2 gene segment and the         unrearranged human TRBJ2-3 gene segment,     -   an unrearranged human TRBJ2-3 gene segment, an unrearranged         human TRBJ2-4 gene segment, and a mouse TCRB non-coding sequence         between the unrearranged human TRBJ2-3 gene segment and the         unrearranged human TRBJ2-4 gene segment,     -   an unrearranged human TRBJ2-4 gene segment, an unrearranged         human TRBJ2-5 gene segment, and a mouse TCRB non-coding sequence         between the unrearranged human TRBJ2-4 gene segment and the         unrearranged human TRBJ2-5 gene segment,     -   an unrearranged human TRBJ2-5 gene segment, an unrearranged         human TRBJ2-6 gene segment, and a mouse TCRB non-coding sequence         between the unrearranged human TRBJ2-5 gene segment and the         unrearranged human TRBJ2-6 gene segment,     -   an unrearranged human TRBJ2-6 gene segment, an unrearranged         human TRBJ2-7 gene segment, and a mouse TCRB non-coding sequence         between the unrearranged human TRBJ2-6 gene segment and the         unrearranged human TRBJ2-7 gene segment, or     -   any combination thereof (e.g., wherein the humanized TCRBDJ1         cluster comprises (i) an unrearranged human TCRBD2 segment         and (ii) any combination of an unrearranged human TRBJ2-1         segment, an unrearranged human TRBJ2-2 segment, an unrearranged         human TCRBJ2-3 segment, an unrearranged human TCRBJ2-4 segment,         an unrearranged human TCRBJ2-5 segment, an unrearranged human         TCRBJ2-6 segment, and an unrearranged human TCRBJ2-7 segment;         and wherein the humanized TCRBDJ2 cluster comprises a mouse         TCRBDJ2 non-coding sequence between the unrearranged human         TCRBD2 segment and any unrearranged human TCRBJ2 segment and a         mouse TCRBDJ2 non-coding sequence between any two consecutive         unrearranged human TCRBJ2 gene segments, optionally wherein the         unrearranged human TCRBD2 and TCRBJ2 gene segments flank the         same mouse TCRBDJ2 non-coding sequences as are normally flanked         by the corresponding mouse tcrbdj2 gene segments).

Methods of making non-human animals include embodiments wherein (a) introducing the first, second and/or third nucleotide sequence(s) encoding the chimeric T cell co-receptor polypeptide(s) into the genome of the non-human animal comprises replacing at an endogenous CD4 locus a nucleotide sequence encoding an endogenous non-human CD4 polypeptide with a nucleotide sequence encoding a chimeric human/non-human CD4 polypeptide, and/or replacing at an endogenous CD8α locus a nucleotide sequence encoding an endogenous non-human CD8α polypeptide with a nucleotide sequence encoding a chimeric human/non-human CD8α polypeptide and replacing at an endogenous CD8β locus a nucleotide sequence encoding an endogenous non-human CD8β polypeptide with a nucleotide sequence encoding a chimeric human/non-human CD8β polypeptide; (b) inserting the unrearranged TCRα locus and/or unrearranged TCRβ locus into the genome of the animal comprises replacing an endogenous non-human TCRα variable gene locus with an unrearranged humanized TCRα variable gene locus comprising at least one human Vα segment and at least one human Jα segment to generate a humanized TCRα variable gene locus, wherein the humanized TCRα variable gene locus is operably linked to endogenous non-human TCRα constant region and/or replacing an endogenous non-human TCRβ variable gene locus with an unrearranged humanized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment to generate a humanized TCRβ variable gene locus, wherein the humanized TCRβ variable gene locus is operably linked to endogenous non-human TCRβ constant region; (c) placing the first, second and/or third nucleic acid sequence(s) encoding chimeric MHC polypeptide(s) into the genome of the non-human animal comprises replacing at an endogenous non-human MHC II locus a nucleotide sequence encoding a non-human MHC II complex with a nucleotide sequence encoding a chimeric human/non-human MHC II complex and replacing at an endogenous non-human MHC I locus a nucleotide sequence encoding a non-human MHC I polypeptide with a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide and/or (d) adding the β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide into the genome of a non-human animal comprises replacing at the endogenous non-human β2 microglobulin locus a nucleotide sequence encoding a non-human β2 microglobulin polypeptide with a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide.

In some embodiments, (a) introducing the first, second and/or third nucleotide sequence into the genome of the non-human animal respectively comprises (i) replacing at an endogenous CD4 locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of an endogenous non-human CD4 polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human CD4 polypeptide in operable linkage with sequences encoding the endogenous non-human CD4 transmembrane and cytoplasmic domains, (ii) replacing at an endogenous CD8α locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of an endogenous non-human CD8α polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human CD8α polypeptide in operable linkage with sequences encoding the endogenous non-human CD8α transmembrane and cytoplasmic domains and/or (iii) replacing at an endogenous CD8β locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of an endogenous non-human CD8β polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human CD8β polypeptide in operable linkage with sequences encoding the endogenous non-human CD8β transmembrane and cytoplasmic domains; (b) inserting the unrearranged TCRα locus and/or unrearranged TCRβ locus into the genome of the animal respectively comprises (i) replacing an endogenous non-human TCRα variable gene locus with an unrearranged humanized TCRα variable gene locus comprising at least one human Vα segment and at least one human Jα segment to generate a humanized TCRα variable gene locus, wherein the humanized TCRα variable gene locus is operably linked to endogenous non-human TCRα constant region and/or (ii) replacing an endogenous non-human TCRβ variable gene locus with an unrearranged humanized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment to generate a humanized TCRβ variable gene locus, wherein the humanized TCRβ variable gene locus is operably linked to endogenous non-human TCRβ constant region; (c) placing the first, second and/or third nucleic acid sequence into the genome of the non-human animal respectively comprises (i) replacing at an endogenous non-human MHC II α locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of a non-human MHC II α polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human HLA class II α polypeptide in operable linkage with sequences encoding the endogenous non-human MHC II α transmembrane and cytoplasmic domains, (ii) replacing at an endogenous non-human MHC II β locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of a non-human MHC II β polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human HLA class II β polypeptide in operable linkage with sequences encoding the endogenous non-human MHC II β transmembrane and cytoplasmic domains and/or (iii) replacing at an endogenous non-human MHC I locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of a non-human MHC I polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human HLA class I polypeptide in operable linkage with sequences encoding the endogenous non-human MHC I transmembrane and cytoplasmic domains; and/or replacing at an endogenous β2 microglobulin locus a nucleotide sequence set forth in exon 2-exon 4 with a nucleotide sequence comprising exons 2, 3, and 4 of a human β2 microglobulin gene.

In one embodiment, the introducing step comprises replacing in a first non-human animal at an endogenous CD4 locus a nucleotide sequence encoding an endogenous non-human CD4 polypeptide with a nucleotide sequence encoding a chimeric human/non-human CD4 polypeptide, replacing in a second non-human animal at an endogenous CD8α locus a nucleotide sequence encoding an endogenous non-human CD8α polypeptide with a nucleotide sequence encoding a chimeric human/non-human CD8α polypeptide and replacing at an endogenous CD8β locus a nucleotide sequence encoding an endogenous non-human CD8β polypeptide with a nucleotide sequence encoding a chimeric human/non-human CD8β polypeptide. In some embodiments, the introducing step comprises replacing in a first non-human animal at an endogenous CD4 locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of an endogenous non-human CD4 polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human CD4 polypeptide in operable linkage with sequences encoding the endogenous non-human CD4 transmembrane and cytoplasmic domains, replacing in a second non-human animal at an endogenous CD8α locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of an endogenous non-human CD8α polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human CD8α polypeptide in operable linkage with sequences encoding the endogenous non-human CD8α transmembrane and cytoplasmic domains and replacing at an endogenous CD8β locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of an endogenous non-human CD8β polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human CD8β polypeptide in operable linkage with sequences encoding the endogenous non-human CD8β transmembrane and cytoplasmic domains. In some embodiments, the replacing steps are performed simultaneously or in any order.

In some embodiments, the inserting step comprises replacing in a third non-human animal an endogenous non-human TCRα variable gene locus with an unrearranged humanized TCRα variable gene locus comprising at least one human Vα segment and at least one human Jα segment to generate a humanized TCRα variable gene locus, wherein the humanized TCRα variable gene locus is operably linked to endogenous non-human TCRα constant region; replacing in a fourth non-human animal an endogenous non-human TCRβ variable gene locus with an unrearranged humanized TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment to generate a humanized TCRβ variable gene locus, wherein the humanized TCRβ variable gene locus is operably linked to endogenous non-human TCRβ constant region. In some embodiments, the replacing steps are performed simultaneously or in any order.

In some embodiments, the placing step comprises, in no particular order, replacing in a fifth non-human animal at an endogenous non-human MHC II locus one or more nucleotide sequence encoding a non-human MHC II complex with one or more nucleotide sequence encoding a chimeric human/non-human MHC II complex; and replacing in the fifth non-human animal at an endogenous non-human MHC I locus a nucleotide sequence encoding a non-human MHC I polypeptide with a nucleotide sequence encoding a chimeric human/non-human MHC I polypeptide. In some embodiments, the placing step comprises replacing in a fifth non-human animal at an endogenous non-human MHC II α locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of a non-human MHC II α polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human MHC II α polypeptide in operable linkage with sequences encoding the endogenous non-human MHC II α transmembrane and cytoplasmic domains and replacing at an endogenous non-human MHC II β locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of a non-human MHC II β polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human MHC II polypeptide in operable linkage with sequences encoding the endogenous non-human MHC II β transmembrane and cytoplasmic domains; and replacing at an endogenous non-human MHC I locus a nucleotide sequence encoding the extracellular portion (or a part thereof) of a non-human MHC I polypeptide with a nucleotide sequence encoding the extracellular portion (or a part thereof) of a human MHC I polypeptide in operable linkage with sequences encoding the endogenous non-human MHC I transmembrane and cytoplasmic domains in the fifth non-human animal. In some embodiments, the replacing steps are performed simultaneously or in any order.

In some embodiments, the adding step comprises replacing in a sixth non-human animal at the endogenous non-human β2 microglobulin locus a nucleotide sequence encoding a non-human β2 microglobulin polypeptide with a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In some embodiments, the human or humanized β2 microglobulin polypeptide is encoded by the nucleotide sequence set forth in exon 2, exon 3, and exon 4 of the human β2 microglobulin gene.

Methods disclosed herein include embodiments wherein a first, second, and/or third nucleotide sequence(s) encoding chimeric T cell co receptor polypeptide(s) is introduced; the TCRα locus and/or unrearranged TCRβ locus is inserted; first, second and/or third nucleic acid sequence(s) encoding chimeric MHC polypeptide(s) is placed; and/or the β2 microglobulin locus is added by breeding a non-human animal comprising one or more of the genetic modifications as described herein to another (or more) non-human animal(s) of the same species comprising the remaining genetic modifications. A non-limiting embodiment includes breeding, in any order, the first, second, third, fourth, fifth and sixth non-human animals as described above.

Methods disclosed herein may comprise homologous recombination in non-human embryonic stem (ES) cells. Methods disclosed herein may be used to generate mice as disclosed herein. Non-human animals expressing chimeric human/non-human CD4, CD8α and/or CD8β T cell co-receptor polypeptides, human(ized) TCR α/β proteins, and chimeric MHC II complex and MHC I (with human or humanized β2 microglobulin) may be generated by (a) first introducing each individual human(ized) gene by homologous recombination in individual ES cells respectively and generating each individual non-human animal from such ES cells, and subsequent breeding of each generated non-human animal in any order, (b) introducing all human(ized) genes by sequential homologous recombination in a single ES cell and then generating a non-human animal from such ES cell, or (c) a combination of sequential homologous recombination at some loci in ES cells and breeding. Animals as disclosed herein may also be generated by breeding the progeny of the initial breeding with other animals as appropriate. Breeding and/or homologous recombination may be accomplished in any preferred order.

Also described herein are targeting vectors, e.g., for use in described methods. In some embodiments, a targeting vector may comprise 5′ and 3′ homology arms for targeting a mouse TCRBDJ region, an unrearranged human TCRBD segment, an unrearranged human TCRBJ segment, and a mouse TRCBDJ non-coding sequence. In some embodiments, (A) the unrearranged human TCRBD segment comprises a sequence set forth at the following human genomic coordinates on chromosome 7 (GRCh38 assembly): 142,786,213-142,786,224, and/or 142,796,365-142,796,414; (B) the unrearranged human TCRBJ segment comprises a sequence set forth at the following human genomic coordinates on chromosome 7 (GRCh38 assembly): 142,786,880-142,786,927; 142,787,017-142,787,064; 142,787,630-142,787,679; 142,788,225-142,788,275; 142,788,498-142,788,547; 142,788,988-142,789,040; 142,795,686-142,795,740; 142,796,560-142,796,610; 142,796,847-142,796,895; 142,796,998-142,797,047; 142,797,119-142,797,166; 142,797,239-142,797,291, and/or 142,797,456-142,797,502; and (C) the mouse TCRBDJ non-coding sequence comprises a mouse TCRBDJ non-coding sequence found between the mouse TCRBD gene segment that is orthologous to the human TCRBD gene segment of (A) and the TCRBJ gene segment that is orthologous to the human TCRBJ gene segment of (B).

Also described are mouse genomes or mouse cells (e.g., ES cells, germ cells, etc) comprising the targeting vector described herein.

Also provided are methods of isolating human TCR variable domains specific for an antigen from a non-human animal comprising isolating from a non-human animal provided herein or made according to a method disclosed herein a T cell or TCR protein that binds to the antigen. In some embodiments, the methods may further comprise identifying a first and/or second nucleic acid encoding the TCRα and/or TCRβ variable domains that binds to the antigen and/or culturing a cell comprising one or more vectors in sufficient conditions for expression of the vector(s), wherein the vector(s) comprises a third and/or fourth nucleic acid respectively identical to or substantially identical to the first and/or second nucleic acids, and wherein the third and/or fourth nucleic acid is cloned in-frame with, e.g., a human TCR constant region gene, e.g., a TCRα constant region gene and/or TCRβ constant region gene, respectively. Tissues and cells comprising the genetic modifications as disclosed herein (which may include rearranged human TCRα and/or TCRβ variable region genes), and nucleic acids encoding such human TCR variable domains expressed by such tissues or cells isolated from a non-human animal modified as described herein are also provided. Also included are (1) recombinant nucleic acids, e.g., expression vectors, comprising the nucleic acid sequences encoding a human TCR variable domain as disclosed herein, e.g., a human rearranged TCRα or human rearranged TCRβ variable region gene, cloned in-frame to an appropriate human TCR constant region gene, e.g., a TCRα constant region gene or TCRβ constant region gene, respectively, (2) host cells comprising such nucleic acids (e.g., expression vectors) and (3) the TCR expressed by the host cells. In some embodiments, recombinant nucleic acids provided herein comprise a human rearranged TCRδ variable region gene or a TCRγ variable region gene, e.g., derived from a non-human animal genetically modified as disclosed herein or a tissue isolated therefrom, cloned in-frame with a human TCRδ constant region gene or a TCRγ constant region gene, respectively.

A method of generating a humanized T cell response in a non-human animal is also provided, the method generally comprising immunizing a non-human animal a non-human animal genetically modified or having a substantially humanized T cell immune system as described herein with an antigen, e.g., a human antigen, e.g., a human tumor antigen, a human bacterial pathogen, a human viral pathogen, etc. In some embodiments, the non-human animal immunized expresses at least 50% of all functional human TCRVα gene segments and/or at least 50% of all functional human TCRVβ gene segments and/or comprises all or substantially all functional human TCRVα gene segments and/or all or substantially all functional human TCRVβ gene segments.

Also provided are in vitro methods of isolating human TCR specific for an antigen, which generally comprise detecting activation of a first cell of a non-human animal after (a) contact with a second cell of a non-human animal and (b) incubation with the antigen; wherein the first cell expresses a chimeric human/non-human T cell co-receptor and either or both (i) a chimeric human/non-human TCRα chain and (ii) a chimeric human/non-human TCRβ chain, and wherein the second cell expresses a chimeric human/non-human MHC polypeptide. The methods may further comprise isolating a TCR from the first cell, or nucleic acids encoding same.

In the in vitro methods disclosed herein, the antigen may be tumor antigen, a viral antigen, an autoantigen, or a bacterial antigen. In some embodiments, the non-human animal is a rodent, e.g., a rat or a mouse. Also provided herein is tissue, a T cell, a TCR (e.g., a soluble TCR), or a nucleic acid encoding all or part of the TCR that is isolated from a non-human animal genetically modified or having a substantially humanized T cell immune system as described herein, a hybridoma or quadroma derived from such a T cell.

Also provided are compositions, e.g., comprising a first and second cell of a non-human animal; wherein the first cell expresses a chimeric human/non-human T cell co-receptor and optionally, either or both (i) a chimeric human/non-human TCRα chain and (ii) a chimeric human/non-human TCRβ chain, and wherein the second cell expresses a chimeric human/non-human MHC polypeptide that associates with the chimeric human/non-human T cell co-receptor. In some embodiments, the first cell is a non-human T cell. In other embodiments, the second cell is a non-human antigen presenting cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation (not to scale) of humanized T cell receptor complex comprising humanized TCR alpha and beta proteins, humanized MHC Class I complexed with humanized β2 microglobulin, and humanized CD8 heterodimer (left panel); as well as T cell receptor complex comprising humanized TCR alpha and beta proteins, humanized MHC Class II heterodimer, and humanized CD4 (right panel). Antigen presented by humanized MHC is depicted as a circle. Mouse regions are depicted as filled shapes while human regions are depicted as striped shapes.

FIGS. 2A-C provide a schematic representation (not to scale) of exemplary chimeric MHC I and MHC II loci, e.g., chimeric HLA-A2/H-2K locus (FIG. 2A), chimeric HLA-DR2/H-2E locus (FIG. 2B), and humanized β2M locus (FIG. 2C). Unless otherwise indicated, human sequences are depicted as empty shapes and mouse sequences are depicted as filled shapes. The striped shape represents exon 1 of H-2E derived from a different mouse strain than the endogenous locus (see Example 1.3 and FIG. 3B). Floxed neomycin phosphotransferase cassette(s) are depicted with arrows labeled accordingly.

FIGS. 3A-C depicts a strategy for generating a humanized MHC locus comprising humanized MHC I and MHC II genes. In the particular embodiment depicted in FIG. 3A, the MHC locus of the generated mouse comprises chimeric HLA-A2/H-2K and HLA-DR2/H-2E sequences (H2-K^(+/1666) MHC-II^(+/6112)) and lacks H2-D sequence (H2-D^(+/delete)) and H-2A sequence (the genetic engineering scheme also results in a deletion of H-2A, see Example 1.2). Large Targeting Vectors (LTVECs) or Cre recombinase construct introduced into ES cells at each stage of humanization are depicted to the right of the arrows. MAID or 4 digit numbers refer to modified allele ID number. FIG. 3B is a schematic diagram (not to scale) of an exemplary HLA-DR2/H-2E large targeting vector. Unless otherwise indicated, human sequences are depicted as empty shapes and mouse sequences are depicted as filled shapes. The striped shape represents exon 1 of H-2E derived from a different mouse strain than the endogenous locus (see Example 1.3). A floxed hygromycin cassette is depicted as an arrow labeled accordingly. FIG. 3C is a schematic representation (not to scale) of exemplary genotypes of chimeric human/mouse MHC loci (** represents H-2L gene that is not present in all mouse strains, e.g., is not present in C57BL/6 or 129 mouse strains), where endogenous mouse H-2K and H-2E loci are respectively replaced by chimeric human/mouse HLA-A2/H-2K and HLA-DR2/H-2E loci (striped shapes), H-2A and H-2D loci were deleted (empty shapes outlined with dotted lines), and remaining loci are endogenous mouse genes (solid shapes outlined with solid lines).

FIG. 4A depicts (not to scale) a progressive strategy for humanization of the mouse TCRα locus, wherein TCRα variable region gene segments are sequentially added upstream of an initial humanization of a deleted mouse locus (MAID1540). Mouse sequence is indicated by filled shapes; human sequence is indicated by empty shapes. MAID refers to modified allele ID number. TRAV=TCR Vα segment, TRAJ=TCR Jα segment (hTRAJ=human TRAJ), TRAC=TCR Ca domain, TCRD=TCRδ. Although not depicted in this figure, each non-coding sequence between each human TRAV and each human TRAJ is human. FIG. 4B depicts (not to scale) a progressive strategy for humanization of the mouse TCRβ locus, wherein TCRβ variable region gene segments are sequentially added to a deleted mouse TCRβ variable locus. Mouse sequence is indicated by filled shapes; human sequence is indicated by empty shapes. MAID refers to modified allele ID number. TRBV or TCRBV=TCRβ V segment. Although not depicted in this figure, each non-coding sequence between each human TRBV, between the human D1 and the closest human J, between the human D2 and each human J, and between each J, is human. FIG. 4C depicts a schematic representation (not to scale) of (a) a TRBDJ1 cluster where the D1 and J1 gene segments are human and the non-coding sequences between them, including RSSs and other intergenic sequences, are mouse, (b) a mouse TRB C1 constant gene, (c) a TRBDJ2 cluster where the D2 and J2 gene segments are human and the non-coding sequences between them, including RSSs and other intergenic sequences, are mouse, and (d) a mouse TRB C2 constant gene. Mouse sequence is indicated by filled shapes; human sequence is indicated by empty shapes. As depicted in this figure, each non-coding sequence between each human TRBD segment and each human TRBJ segment, and between each TRBJ segment, is mouse. A flox sequence is depicted as an arrow labeled accordingly

FIG. 5A depicts a schematic representation (not to scale) of the chimeric CD4 locus. Human coding exons are presented by striped shapes, mouse coding exons are presented by filled shapes, and non-coding exons are presented by empty shapes. Immunoglobulin-like domains (Ig), transmembrane (TM), cytoplasmic (CYT) and signal peptide (Signal) coding exons, as well as 3′ untranslated regions (UTR), are indicated. A floxed (loxP) neomycin phosphotransferase (Pgk-neo) cassette is depicted with arrows labeled accordingly. FIG. 5B depicts a schematic representation (not to scale) of the chimeric CD8a and CD8b loci. Human coding exons are presented by striped shapes, mouse coding exons are presented by filled shapes, and non-coding exons are presented by empty shapes. Immunoglobulin-like domains (IgV), transmembrane (TM), cytoplasmic (CYT) and signal peptide (Signal) coding exons, as well as 3′ untranslated regions (UTR), are indicated. Floxed (loxP) hygromycin (Hyg) and neomycin phosphotransferase (Pgk-neo) cassettes are depicted with arrows labeled accordingly.

FIGS. 6A-C are FACS contour plots of thymic cells isolated from a control mouse or a mouse comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci, gated on singlets, and stained with (FIG. 6A) anti-mouse CD19 and anti-mouse CD3 antibodies, (FIG. 6B) anti-mouse CD19 and anti-mouse F4/80 antibodies, or (FIG. 6C) anti-mouse CD8α and anti-mouse CD4 antibodies (left panel) or anti-human CD8α and anti-human CD4 antibodies (right panel). In this figure, the TM I/II B C4/8 mouse comprises fully human TCRBDJ1 and TCRBJ2 clusters.

FIGS. 7A-G are FACS contour plots of thymic cells isolated from a control mouse or a mouse comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci, gated on CD19+ cells, F4/80+ cells or CD3+ cells, and stained with (FIGS. 7A, 7B) anti-human B2M or anti-mouse H-2D antibodies; (FIGS. 7C, 7D) anti-HLA-A2 or anti-HLA-DR antibodies; (FIGS. 7E, 7F) anti-H-2D and anti-I^(A)I^(E) antibodies; or (FIG. 7G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8α antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom). In this figure, the TM I/II B C4/8 mouse comprises fully human TCRBDJ1 and TCRBJ2 clusters.

FIG. 8 provides FACS contour plots of thymic cells isolated from a control mouse or a mouse comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8), gated on CD3⁺CD4⁺ cells, and stained with anti-mouse FoxP3 and anti-mouse CD25 antibodies. In this figure, the TM I/II B C4/8 mouse comprises fully human TCRBDJ1 and TCRBJ2 clusters.

FIGS. 9A-E are FACS contour plots of splenic cells isolated from a control mouse or a mouse comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci, gated on singlets, CD3+ cells, CD4+ T cells, or CD8+ T cells, and stained with (FIG. 9A) anti-mouse CD19 and anti-mouse CD3, (FIG. 9B) anti-mouse CD19 and anti-mouse F4/80 antibodies, (FIG. 9C) anti-mouse CD4 and anti-mouse CD8α antibodies (left) or anti-human CD4 and anti-human CD8α antibodies (right), or (FIGS. 9D, 9E) anti-mouse CD44 and anti-mouse CD62L antibodies. In this figure, the TM I/II B C4/8 mouse comprises fully human TCRBDJ1 and TCRBJ2 clusters.

FIGS. 10A-G are FACS contour plots of splenic cells isolated from a control mouse or a mouse comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci, gated on CD19+ cells, F4/80+ cells, or CD3⁺ cells, and stained with (FIGS. 10A, 10B) anti-human B2M or anti-mouse H-2D antibodies, (FIGS. 10C, 10D) anti-HLA-A2 or anti-HLA-DR antibodies, (FIGS. 10E, 10F) anti-H-2D and anti-I^(A)I^(E) antibodies, or (FIG. 10G) anti-mouse CD4 and anti-human CD4 antibodies (top), anti-mouse CD8α and anti-human CD8α antibodies (middle), and anti-mouse CD8β and anti-human CD8β antibodies (bottom). In this figure, the TM I/II B C4/8 mouse comprises fully human TCRBDJ1 and TCRBJ2 clusters.

FIG. 11 provides FACS contour plots of splenic cells isolated from a control mouse or a mouse comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8), gated on CD3⁺CD4⁺ cells, and stained with anti-mouse FoxP3 and anti-mouse CD25 antibodies. In this figure, the TM I/II B C4/8 mouse comprises fully human TCRBDJ1 and TCRBJ2 clusters.

FIG. 12A provides the number of splenic cells (spots per well (Mean+SD); y-axis) that produce IFN-γ in an enzyme-linked immunosorbent spot assay after isolation from a control mouse or a mouse comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci and incubation in the absence of peptide (200k cells only; x-axis) or presence of 10 μg/ml or 1 μg/ml MAGE-A3 peptide (x-axis). In FIGS. 12B-12C, WT and TM I/II B C4/8 mice were immunized with HLA-A2-restricted NY-ESO-1₁₅₇₋₁₆₅ or MAGE-A3₂₇₁₋₂₇₉ peptide, respectively. Day 14 post-immunization, the presence of antigen-specific T cells was quantified by IFN-γ Elispot in pooled spleen and lymph node (LN) cells cultured in the presence of antigenic peptide (10 mg/ml) or no peptide. In FIG. 12B, one naïve (no immunization) and two immunized mice of each genotype were analyzed. In FIG. 12C, two naïve and four immunized mice of each genotype were analyzed. FIG. 12D provides an illustrative Jurkat-based reporter system developed to test TCR-mediated signaling in response to peptide-pulsed, engineered antigen presenting cells (APCs), consisting of 293T cells expressing human CD80 and CD86. Parental reporter cells lacking surface TCR (JRT3/hCD8/hCD28/AP1.luciferase) were transduced with lentiviral supernatant encoding cloned TM I/II B C4/8 derived-TCRs to generate reporter JRT3 reporter lines. In FIG. 12E, surface TCR expression was measured on transduced and parental JRT3 lines by flow cytometry. In FIG. 12F, reporter lines expressing indicated TCRs (parental JRT3, #001, #050, #063, #188, #229) were co-cultured with APCs pulsed with or without NY-ESO-1₁₅₇₋₁₆₅ peptide. 5×10⁴ JRT3 cells were incubated with varying numbers of APCs (starting at 3×10⁵ APCs, with 2-fold dilution series; x-axis). After 4 hours, AP1-driven luciferase activity was measured. Relative luminescence units (RLU; y-axis) are plotted for NY-ESO-1 peptide-pulsed cultures. Signal-to-noise (S/N) ratio values were calculated as the ratio of NY-ESO-1 peptide co-cultures to no-peptide cultures (not plotted), using RLU values from the highest APC concentration tested (effector:target [E:T] ratio of 1:6). In FIG. 12G, JRT3-based TCR reporter assays were performed using 293T APCs pulsed with NY-ESO-1₁₅₇₋₁₆₅ or predicted off-target peptides. FIGS. 12H-12I provide illustrations related to expression of TCR isolated from TM I/II B C4/8 mice by targeting to human TRAC locus. FIG. 12H provides a schematic of a productively re-arranged human TRAC locus, with sgRNA cut site and AAV vector for insertion of customized TCRa/b chains. Expression relies on in-frame cassette insertion within the first TRAC exon, with transcription driven by the upstream, endogenous Vα promoter. The portion of the TRAC domain encoded by its first exon (striped region) is included within the right homology arm (HA-R), and the remaining TRAC domain is encoded by endogenous downstream exons. TRAC* and TRBC1* indicate constant domain sequences that have been re-coded to eliminate sgRNA binding while preserving amino acid sequence. In FIG. 12I, primary human T cells were nucleofected with Cas9 RNPs containing a mixture of TRAC/TRBC sgRNA, or a non-targeting sgRNA, followed by transduction with AAV encoding a homology directed repair (HDR) template for insertion of NY-ESO-1 specific TCR050. Expression of total surface TCR (via CD3e) and TCR050 (via pMHC tetramer) were analyzed by flow cytometry 7 days post-transduction. FIGS. 12J and 12K show the anti-tumor activity of a TCR isolated from TM I/II B C4/8 mice. FIG. 12J provides a scheme for expression and analysis of TCR050. FIG. 12K provides expression of NY-ESO specific TCR050 measured in TRAC targeted T cells by NY-ESO-1₁₅₇₋₁₆₅ tetramer (top panels) and Vβ specific antibodies (bottom panels), compared to an irrelevant control TCR (HPV) and untransduced cells. In the bottom panel, TCR050 and HPV TCRs were stained for their cognate Vβ chains. Untransduced cells were stained for Vβ13.6 to establish basal usage in the PBMC donor. FIG. 12L provides the cytotoxic activity against A375 cells as measured in a 2-hour calcein-AM release assay. In FIG. 12M, NSG mice (n=5) were implanted with A375 tumor cells and administered T cells on the same day. Tumor growth was measured over time, with values representing mean (±SEM) tumor volume In FIG. 12N, tumor growth curves for individual mice from FIG. 12M are shown. In FIG. 12O, serum from mice was harvested 3 days after T cell administration and analyzed for human IFNγ. Values represent mean (±SEM) concentrations of IFNγ. ****, p<0.0001 by two-way ANOVA with Tukey test for multiple comparisons. In this figure, the TM I/II B C4/8 mouse comprises fully human TCRBDJ1 and TCRBJ2 clusters.

FIG. 13A depicts progression of acute Armstrong strain viral infection in either control or mice comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci; the timeline for the experiment is depicted at the top of the figure, and measurement of viral titers on various days post-infection for both mouse strains is depicted in the bottom graph. FIG. 13B depicts progression of chronic Clone 13 strain viral infection in either control or mice comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci; the timeline for the experiment is depicted at the top of the figure, and the measurement of viral titers on Day 21 post-infection for both mouse strains is depicted in the bottom graph. T cells from uninfected or chronically infected TM I/II B C4/8 or control B6 mice were stained with anti-PD1, anti-Lag3, and anti-Tim3 antibodies (FIG. 13C; x-axis); the figure provides a quantification of cells staining positive (% positive cells; y-axis). In this figure, the TM I/II B C4/8 mouse comprises fully human TCRBDJ1 and TCRBJ2 clusters.

FIG. 14 depicts progression of chronic Clone 13 strain viral infection in either control or TM I/II B C4/8 mice after prior acute Armstrong strain infection; the timeline for the experiment is depicted at the top of the figure, and measurement of viral titers on Day 31 post-infection is depicted in the bottom graph. Mock infected mice were included in the experiment as an additional control. In this figure, the TM I/II B C4/8 mouse comprises fully human TCRBDJ1 and TCRBJ2 clusters.

FIGS. 15A-B depicts the number of CD8⁺ cells (y-axis; IFN-γ Positive Cells) that produced IFN-γ in response to LCMV peptides that are HLA-A2 restricted (GPC10-18; N69-77; Z49-58), H2D^(b) restricted (GP33-41), ovalbumin, or incubation alone and were isolated from either control animals (FIG. 15A) or mice comprising humanized MHC I, MHC II α and TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci (FIG. 15B), each of which received a mock infection (mock; n=1 each group) or an acute Armstrong strain infection (Arm; n=3 each group). The % of IFNγ+CD8+ lymphocytes (y-axis) after stimulation with the indicated peptides (OVA, GP33, NP69, GPC10, GPC447 or Z49) during a time course of infection (days post infection; x-axis) in mice comprising humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B C4/8) loci or control B6 animals are shown in FIGS. 15C and 15D, respectively. In this figure, the TM I/II B C4/8 mouse comprises fully human TCRBDJ1 and TCRBJ2 clusters.

FIG. 16A provides representative flow cytometry analysis contour plots and FIG. 16B provides cell percentages of mCD19+ B cells, mCD3+ T cells, hCD4+ T cells and hCD8+ T cells in spleens of TM I/II B C4/8 (VelociT) and WT mice (n=4). FIG. 16C provides representative flow cytometry analysis contour plots and FIG. 16D provides cell numbers and proportions for DN, DP, CD4 SP, CD8 SP cells, and developmental DN1, DN2, DN3, and DN4 thymocytes in VelociT and WT mice (n=4). In this figure, VelociT=the TM I/II B C4/8 mouse comprising humanized TCRBDJ1 and TCRBJ2 clusters comprising mouse TCRBDJ1 and TCRBDJ2 non-coding sequences and human TCRBDJ1 and TCRBDJ2 coding sequences; WT=wildtype control mice.

FIGS. 17A-C provides lymphocyte distribution and phenotyping in central and peripheral compartments for TM I/II B C4/8 mice. FIG. 17A provides total numbers of pan CD3+ T cells, helper CD4+ T cells, cytotoxic CD8+ T cells, and CD19+ B cells in spleen (n=4, mean+/−SD) of VelociT and WT mice. FIG. 17B provides serum IgG and IgM levels of VelociT and WT mice. FIG. 17C provides representative flow cytometry contour plots and percentages of CD3+CD4+ and CD3+CD8+ naïve, central memory, and effector memory T cell subsets in spleen (n=4). In this figure, VelociT=the TM I/II B C4/8 mouse comprising humanized TCRBDJ1 and TCRBJ2 clusters comprising mouse TCRBDJ1 and TCRBDJ2 non-coding sequences and human TCRBDJ1 and TCRBDJ2 coding sequences; WT=wildtype control mice.

FIG. 18A-B provide data showing TM I/II B C4/8 mice develop Tregs. FIG. 18A depicts representative flow cytometry analyses for CD3+ CD4+ FoxP3+ Tregs and FIG. 18B depicts percentages of Tregs in spleen (upper panel) and thymus (lower panel) from VelociT and WT mice (n=4). In this figure, VelociT=the TM I/II B C4/8 mouse comprising humanized TCRBDJ1 and TCRBJ2 clusters comprising mouse TCRBDJ1 and TCRBDJ2 non-coding sequences and human TCRBDJ1 and TCRBDJ2 coding sequences; WT=wildtype control mice.

FIGS. 19A-19B provides data showing the presence of NK cells in TM I/II B C4/8 mice. FIG. 19A provides the flow cytometry gating strategy for detecting NK (CD19-CD3-NKp46+) and NKT (CD19−CD3+NKp46+) populations in the spleen of VelociT and WT mice and FIG. 19B provides the percentages and total counts for all VelociT or WT mice (n=4). In this figure, VelociT=the TM I/II B C4/8 mouse comprising humanized TCRBDJ1 and TCRBJ2 clusters comprising mouse TCRBDJ1 and TCRBDJ2 non-coding sequences and human TCRBDJ1 and TCRBDJ2 coding sequences; WT=wildtype control mice.

FIGS. 20A-D(i-ii) provide data showing normal myeloid and antigen presenting cells in TM I/II B C4/8 mice. FIG. 20A provides percentages and numbers of total of indicated myeloid cell populations in spleens of VelociT and WT mice (n=4). FIG. 20B provides human MHC I and II molecule surface expression on CD19+ B cells. FIG. 20C provides human MHC I expression on kidney epithelial cells (KECs)−/+mouse IFN-γ treatment. FIG. 20D illustrates a preserved myeloid cell compartment and provides the gating strategies defining the myeloid populations in the spleens of (FIG. 20Di) WT mice or (FIG. 20Dii) VelociT mice. In this figure, VelociT=the TM I/II B C4/8 mouse comprising humanized TCRBDJ1 and TCRBJ2 clusters comprising mouse TCRBDJ1 and TCRBDJ2 non-coding sequences and human TCRBDJ1 and TCRBDJ2 coding sequences; WT=wildtype control mice.

FIGS. 21A-D provides the V and J gene usage frequencies in the TCRβ (FIG. 21A, FIG. 21B) and TCRα (FIG. 21C, FIG. 21D) repertoires of naïve CD4⁺ splenic T cells in TM I/II B C4/8 mice comprising mouse TCRBDJ1 and TCRBDJ2 non-coding sequences and human TCRBDJ1 and TCRBDJ2 coding sequences (N=3). The gene segments were arranged left to right on the x axis according to their position on the human chromosome from distal to proximal relative to their constant regions. Error bars represent SEM across individual samples.

FIG. 22A-D provides TM I/II B C4/8 CD8 responses to acute and chronic LCMV infection. In FIG. 22A, TM I/II B C4/8 and B6 mice were infected with LCMV Armstrong (2e5 FFU, IP), sacrificed at indicated timepoints, and spleens analyzed for LCMV virus titers by fluorescent focus assay (FFA). In FIG. 22B, Chronic infection was established in TM I/II B C4/8 and B6 mice by infection with LCMV CL13 (5e6 FFU, IV). Mice were sacrificed d21 post infection, and spleens analyzed for LCMV virus titers by FFA. In FIG. 22C, CD8+ T cells from d21 CL13 infected and naïve TM I/II B C4/8 mice were analyzed by flow cytometry for exhaustion markers PD1, LAG3, and TIM3. In FIG. 22D, LCMV immune mice (LCMV Armstrong 2e5 FFU, IP) were re-challenged 17 days post primary infection with a high dose of LCMV CL13 (5e6 FFU, IV). Spleens were analyzed for LCMV titers by FFA d14 post CL13 challenge. In this figure, the TM I/II B C4/8 mouse comprises humanized TCRBDJ1 and TCRBJ2 clusters comprising mouse TCRBDJ1 and TCRBDJ2 non-coding sequences and human TCRBDJ1 and TCRBDJ2 coding sequences.

FIGS. 23A-B show TM I/II B C4/8 mice generate CD8 T cell responses to LCMV. In FIG. 23A, CD8+ T cells from LCMV Armstrong infected VelociT and B6 WT controls were analyzed by IFN-γ ICS for reactivity to control and LCMV-specific CD8 peptides. Shown are representative flow cytometric IFNγ ICS analyses of splenocytes from either a d14 LCMV infected VelociT mouse (top) or B6 mouse (bottom) stimulated with either LCMV HLA-A2 Z49 or H2db GP33 peptides, respectively. FIG. 23B provides a summary of peptide reactivities of CD8+ T cells from VelociT mice (top) and B6 mice (bottom) at indicated timepoints (n=3-5 mice/day) for H2Kb ova257, H2db LCMV GP33, HLA-A2 LCMV NP69, HLA-A2 GPC10, HLA-A2 GPC447, and HLA-A2 Z49. In this figure, VelociT=the TM I/II B C4/8 mouse comprising humanized TCRBDJ1 and TCRBJ2 clusters comprising mouse TCRBDJ1 and TCRBDJ2 non-coding sequences and human TCRBDJ1 and TCRBDJ2 coding sequences; WT=wildtype control mouse.

FIGS. 24A-C show TM I/II B C4/8 mice develop EAE in response to MOG₃₅₋₅₅ peptide immunization. FIG. 24A provides clinical EAE scores of C57Bl/6 mice and VelociT mice immunized with MOG₃₅₋₅₅ peptide emulsified in CFA. FIG. 24B and FIG. 24C provide data from IFN-γ and IL-17A ELISspot assays, respectively, from splenocytes of MOG₃₅₋₅₅ immunized mice cultured in the presence of 10 μg/ml of MOG₃₅₋₅₅ peptide or vehicle DMSO for 24 h (FIG. 24B) or 48 h (FIG. 24C). Data are presented as mean±S.E.M (A) and mean±S.D of spot-forming cells from splenocytes of individual mice, obtained from culture triplicates (B-C). For all graphs, n=6-7 mice from one experiment for 057B1/6 mice and VelociT mice, respectively. In this figure, VelociT=the TM I/II B C4/8 mouse comprising humanized TCRBDJ1 and TCRBJ2 clusters comprising mouse TCRBDJ1 and TCRBDJ2 non-coding sequences and human TCRBDJ1 and TCRBDJ2 coding sequences.

DETAILED DESCRIPTION

Disclosed herein are non-human animals (e.g., rodents, e.g., mice or rats) genetically engineered to express a humanized T cell co-receptor (e.g., humanized CD4 and/or CD8 (e.g., CD8α and/or CD8β)), a human or humanized major histocompatibility complex (MHC) that binds the humanized T cell co-receptor (e.g., human or humanized MHC II (e.g., MHC II α and/or MHC II β chains) and/or MHC I (e.g., MHC Iα), and optionally human or humanized β2 microglobulin) and/or a human or humanized T cell receptor (TCR), as well as embryos, tissues, and cells expressing the same. The development of the cellular arm of the immune system of the non-human animals disclosed herein is comparable to control animals, e.g., the thymus and spleen comprises similar absolute numbers of thymocytes and CD3+ cells. This is in stark contrast to other non-human animals modified to comprise both human TCR (α and β) and a chimeric human/mouse MHC I molecule, see, e.g., Li (2010) Nature Medicine 16:1029-1035 and supplementary materials. Such animals showed a decrease in T cell populations compared not only to wildtype control animals, but also animals modified with only human TCR, and animals modified with only the chimeric human/mouse MHC I molecule, id. Accordingly, provided herein are non-human animals engineered to co-express a humanized CD4 co-receptor and a humanized MHC II and/or a humanized CD8 co-receptor and a humanized MHC I, and optionally a humanized TCR. Methods for making a genetically engineered animal that expresses at least one humanized T cell co-receptor (e.g., humanized CD4 and/or CD8), at least one humanized MHC that associates with the humanized T cell co-receptor (e.g., humanized MHC II and/or MHC I that associate with humanized CD4 and/or CD8, respectively) and/or the humanized TCR are also provided. Methods for using the genetically engineered animals that mount a substantially humanized T cell immune response for developing human therapeutics are also provided.

Substantially Humanized T Cell Immune Responses

Disclosed herein are non-human animals that are genetically modified to mount substantially humanized T cell immune responses. The mice disclosed herein express at least one human or humanized T cell co-receptor, at least one human or humanized major histocompatibility complex (MHC) capable of associating with the at least one human or humanized T cell co-receptor, and/or a human or humanized T cell receptor (TCR), which is preferably capable of recognizing an antigen presented in the context of human or humanized MHC in association with a human or humanized T cell co-receptor and providing activation signals to the non-human cell, e.g., non-human T cell, expressing the human or humanized TCR. The human or humanized T cell co-receptor, human or humanized TCR and/or human or humanized MHC may be encoded by the genome of the non-human animal. In preferred embodiments, upon immunization with an antigen, the non-human animals present HLA restricted epitopes of the antigen to TCR derived from human TCR gene segments, e.g., a human TCRα V segment, a human TCRα J segment, a human TCRβ V segment, human TCRβ D segment and/or a human TCRβJ segment.

Accordingly, encompassed by the invention is a genetically modified non-human animal whose genome comprises (e.g., at an endogenous locus) a nucleotide sequence encoding a humanized T cell co-receptor polypeptide (e.g., CD4 or CD8 polypeptide), wherein the chimeric T cell co-receptor polypeptide comprises conservative amino acid substitutions of the amino acid sequence(s) described herein and/or a nucleic acid sequence encoding a humanized MHC polypeptide that associates with the humanized T cell co-receptor polypeptide, wherein the humanized MHC polypeptide comprises conservative amino acid substitutions of the amino acid sequence(s) described herein.

A conservative amino acid substitution includes substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Conservative amino acid substitutions may be achieved by modifying a nucleotide sequence so as to introduce a nucleotide change that will encode the conservative substitution. In general, a conservative amino acid substitution will not substantially change the functional properties of interest of a protein, for example, the ability of CD4 or CD8 to associate with, e.g., bind to MHC II or MHC I, respectively, and may, e.g., increase sensitivity of TCR to MHC-presented antigen. Examples of groups of amino acids that have side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and, sulfur-containing side chains such as cysteine and methionine. Conservative amino acids substitution groups include, for example, valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, glutamate/aspartate, and asparagine/glutamine. In some embodiments, a conservative amino acid substitution can be a substitution of any native residue in a protein with alanine, as used in, for example, alanine scanning mutagenesis. In some embodiments, a conservative substitution is made that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. ((1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256:1443-45), hereby incorporated by reference. In some embodiments, the substitution is a moderately conservative substitution wherein the substitution has a nonnegative value in the PAM250 log-likelihood matrix.

One skilled in the art would understand that in addition to the nucleic acid residues encoding humanized T cell co-receptor polypeptides, humanized MHC polypeptides, and/or TCR variable regions described herein, due to the degeneracy of the genetic code, other nucleic acids may encode the polypeptides of the invention. Therefore, in addition to a genetically modified non-human animal that comprises in its genome a nucleotide sequence encoding a humanized T cell co-receptor polypeptide (e.g., CD4 or CD8 polypeptide), an unrearranged T cell receptor variable gene locus (e.g., TCRα and/or TCRβ) comprising human unrearranged gene segments, and/or a nucleic acid sequence encoding a humanized MHC polypeptide capable of associating with the humanized T cell co-receptor polypeptide with conservative amino acid substitutions, also provided is a non-human animal whose genome comprises a nucleotide sequence encoding a humanized T cell co-receptor polypeptide (e.g., CD4 or CD8 polypeptide), an unrearranged T cell receptor variable gene locus (e.g., TCRα and/or TCRβ) comprising human unrearranged gene segments, and/or a nucleic acid sequence encoding a humanized MHC polypeptide capable of associating with the humanized T cell co-receptor polypeptide, which differs from that described herein due to the degeneracy of the genetic code.

The identity of a sequence may be determined by a number of different algorithms known in the art that can be used to measure nucleotide and/or amino acid sequence identity. In some embodiments described herein, identities are determined using a ClustalW v. 1.83 (slow) alignment employing an open gap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnet similarity matrix (MacVector™ 10.0.2, MacVector Inc., 2008). The length of the sequences compared with respect to identity of sequences will depend upon the particular sequences. In various embodiments, identity is determined by comparing the sequence of a mature protein from its N-terminal to its C-terminal. In various embodiments when comparing a chimeric human/non-human sequence to a human sequence, the human portion of the chimeric human/non-human sequence (but not the non-human portion) is used in making a comparison for the purpose of ascertaining a level of identity between a human sequence and a human portion of a chimeric human/non-human sequence (e.g., comparing a human ectodomain of a chimeric human/mouse protein to a human ectodomain of a human protein).

The terms “homology” or “homologous” in reference to sequences, e.g., nucleotide or amino acid sequences, means two sequences which, upon optimal alignment and comparison, are identical in, e.g., at least about 75% of nucleotides or amino acids, e.g., at least about 80% of nucleotides or amino acids, e.g., at least about 90-95% nucleotides or amino acids, e.g., greater than 97% nucleotides or amino acids. One skilled in the art would understand that, for optimal gene targeting, the targeting construct should contain arms homologous to endogenous DNA sequences (i.e., “homology arms”); thus, homologous recombination can occur between the targeting construct and the targeted endogenous sequence.

The term “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. As such, a nucleic acid sequence encoding a protein may be operably linked to regulatory sequences (e.g., promoter, enhancer, silencer sequence, etc.) so as to retain proper transcriptional regulation. In addition, various portions of the chimeric or humanized protein of the invention may be operably linked to retain proper folding, processing, targeting, expression, and other functional properties of the protein in the cell. Unless stated otherwise, various domains of the chimeric or humanized proteins of the invention are operably linked to each other.

The term “replacement” in reference to gene replacement refers to placing exogenous genetic material at an endogenous genetic locus, thereby replacing all or a portion of the endogenous gene with an orthologous or homologous nucleic acid sequence. As demonstrated in the Examples below, in one embodiment, nucleic acid sequences of endogenous loci encoding portions of mouse CD4 or CD8 (CD8α and/or CD8β) polypeptides were replaced by nucleotide sequences encoding portions of human CD4 or CD8 (CD8α and/or CD8β) polypeptides, respectively.

“Functional” as used herein, e.g., in reference to a functional polypeptide, refers to a polypeptide that retains at least one biological activity normally associated with the native protein. For example, in some embodiments of the invention, a replacement at an endogenous locus (e.g., replacement at an endogenous non-human CD4 or CD8 locus) results in a locus that fails to express a functional endogenous polypeptide.

Humanized T Cell Co-Receptor(s)

Disclosed herein are non-human animals that express at least one human or humanized T cell co-receptor, e.g., CD4, CD8α and/or CD8β. Accordingly, a non-human animal as disclosed herein comprises at least one of a first, second, and/or third nucleotide sequence, each of which encodes a different human or chimeric human/non-human T cell co-receptor polypeptide selected from a human or humanized CD4 polypeptide, a human or humanized CD8α polypeptide, and a human or humanized CD8β polypeptide. Use of the first, second, third designations herein is not to be construed as limiting the non-human animals disclosed herein as requiring all three nucleotide sequences or the presence of any of the co-receptor nucleotide sequences in any order. Accordingly, a non-human animal as disclosed herein may comprise a nucleic acid sequence or nucleic acid sequences encoding a human or humanized CD4 and/or a human or humanized CD8 (e.g., human or humanized CD8α and/or CD8β) polypeptide(s).

In one embodiment, a non-human animal as disclosed herein comprises a first nucleotide sequence encoding a human or humanized CD4 polypeptide. In another embodiment, a non-human animal as disclosed herein comprises a first nucleotide sequence encoding a human or humanized CD8α polypeptide and a second nucleotide sequence encoding a human or humanized CD8β polypeptide. In another embodiment, a non-human animal as disclosed herein comprises first and second nucleotide sequences encoding human or humanized CD8α and CD8β polypeptides and further comprises a third nucleotide sequence encoding a human or humanized CD4 polypeptide.

Human or Humanized CD4

In various embodiments, the invention generally provides genetically modified non-human animals that comprise in their genome, e.g., at an endogenous CD4 locus, a nucleotide sequence encoding a human or humanized CD4 polypeptide; thus, the animals express a human or humanized CD4 polypeptide.

Human CD4 gene is localized to chromosome 12, and is thought to contain 10 exons. CD4 gene encodes a protein with amino-terminal hydrophobic signal sequence, encoded by exons 2 and 3 of the gene. The protein comprises four extracellular immunoglobulin-like domains, Ig1-Ig4, also commonly and respectively referred to as D1-D4 domains. Maddon et al. (1987) Structure and expression of the human and mouse T4 genes, Proc. Natl. Acad. Sci. USA 84:9155-59. D1 domain is believed to be encoded by exon 3 (sequence downstream of signal peptide) and exon 4, while D2, D3, and D4 are encoded by a separate exon each—exons 5, 6, and 7, respectively (see FIG. 5A: D1, D2, D3 and D4 domains are encoded by sequences designated as Ig1, Ig2, Ig3 and Ig4, respectively). Littman (1987) The Structure of the CD4 and CD8 Genes, Ann. Rev. Immunol. 5:561-84; Hanna et al. (1994) Specific Expression of the Human CD4 Gene in Mature CD4+CD8− and Immature CD4+CD8+ T cells and in Macrophages of Transgenic Mice, Mol. Cell. Biol. 14(2):1084-94; Maddon et al., supra. At areas of high protein concentration, such as the area of contact between T cell and antigen-presenting cell, the molecule tends to homodimerize through interactions between opposing D4 domains. Zamoyska (1998) CD4 and CD8: modulators of T cell receptor recognition of antigen and of immune responses? Curr. Opin. Immunol. 10:82-87; Wu et al. (1997) Dimeric association and segmental variability in the structure of human CD4, Nature 387:527; Moldovan et al. (2002) CD4 Dimers Constitute the Functional Component Required for T Cell Activation, J. Immunol. 169:6261-68.

D1 domain of CD4 resembles immunoglobulin variable (V) domain, and, together with a portion of D2 domain, is believed to bind (associate with) MHC II, e.g., at an MHC II co-receptor binding site. Huang et al. (1997) Analysis of the contact sites on the CD4 Molecule with Class II MHC Molecule, J. Immunol. 158:216-25. In turn, MHC II interacts with T cell co-receptor CD4 at the hydrophobic crevice at the junction between MHC II α2 and β2 domains. Wang and Reinherz (2002) Structural Basis of T Cell Recognition of Peptides Bound to MHC Molecules, Molecular Immunology, 38:1039-49.

Domains D3 and D4 of the CD4 co-receptor are believed to interact with the TCR-CD3 complex as the substitution of these two domains abrogated the ability of CD4 to bind to TCR. Vignali et al. (1996) The Two Membrane Proximal Domains of CD4 Interact with the T Cell Receptor, J. Exp. Med. 183:2097-2107. CD4 molecule exists as a dimer, and residues in the D4 domain of the molecule are believed to be responsible for CD4 dimerization. Moldovan et al. (2002) CD4 Dimers Constitute the Functional Components Required for T Cell Activation, J. Immunol. 169:6261-68.

Exon 8 of the CD4 gene encodes the transmembrane domain, while the remainder of the gene encodes the cytoplasmic domain. CD4 cytoplasmic domain possesses many distinct functions. For example, the cytoplasmic domain of CD4 recruits a tyrosine kinase Lck. Lck is a Src family kinase that is associated with CD4 and CD8 cytoplasmic domains and simultaneous binding of the co-receptors and TCRs to the same MHC leads to increased tyrosine phosphorylation of CD3 and ζ chain of the TCR complex, which in turn leads to recruitment of other factors that play a role in T cell activation. Itano and colleagues have proposed that cytoplasmic tail of CD4 also promotes differentiation of CD4+CD8+ T cells into CD4+ lineage by designing and testing expression of hybrid protein comprising CD8 extracellular domain and CD4 cytoplasmic tail in transgenic mice. Itano et al. (1996) The Cytoplasmic Domain of CD4 Promotes the Development of CD4 Lineage T Cells, J. Exp. Med. 183:731-41. The expression of the hybrid protein led to the development of MHC I-specific, CD4 lineage T cells. Id.

CD4 co-receptor appears to be the primary receptor for HIV virus, with the CD4+ T cell depletion being an indicator of disease progression. The cytoplasmic tail of CD4 appears to be essential for delivering apoptotic signal to CD4+ T cells in HIV-induced apoptosis. Specifically, the interaction of CD4 and Lck was shown to potentiate HIV-induced apoptosis in these cells. Corbeil et al. (1996) HIV-induced Apoptosis Requires the CD4 Receptor Cytoplasmic Tail and Is Accelerated by Interaction of CD4 with p56lck, J. Exp. Med. 183:39-48.

T cells develop in the thymus progressing from immature CD4−/CD8− (double negative or DN) thymocytes to CD4+/CD8+ (double positive or DP) thymocytes, which eventually undergo positive selection to become either CD4+ or CD8+ (single positive or SP) T cells. DP thymocytes that receive signals through MHC I-restricted TCR differentiate into CD8+ T cells, while DP thymocytes that receive signals through MHC II-restricted TCR differentiate into CD4+ T cells. The cues received by the DP cell that lead to its differentiation into either CD4+ of CD8+ T cell have been a subject of much research. Various models for CD4/CD8 lineage choice have been proposed and are reviewed in Singer et al. (2008) Lineage fate and intense debate: myths, models and mechanisms of CD4-versus CD8-lineage choice, Nat. Rev. Immunol. 8:788-801.

Deactivation of a specific T cell co-receptor as a result of positive selection is a product of transcriptional regulation. For CD4, it has been shown that an enhancer located 13 kb upstream of exon 1 of CD4 upregulates CD4 expression in CD4+ and CD8+ T cells. Killeen et al. (1993) Regulated expression of human CD4 rescues helper T cell development in mice lacking expression of endogenous CD4, EMBO J. 12:1547-53. A cis-acting transcriptional silencer located within the first intron of murine CD4 gene functions to silence expression of CD4 in cells other than CD4+ T cells. Siu et al. (1994) A transcriptional silencer control the developmental expression of the CD4 gene, EMBO J. 13:3570-3579.

Because important transcriptional regulators (e.g., promoters, enhancers, silencers, etc.) that control CD4 lineage choice were missing in several strains of previously developed transgenic mice expressing human CD4, these mice were not able to recapitulate normal T cell lineage development, and produced immune cells other than CD4+ T cells that expressed CD4. See, e.g., Law et al. (1994) Human CD4 Restores Normal T Cell Development and Function in Mice Deficient in CD4, J. Exp. Med. 179:1233-42 (CD4 expression in CD8+ T cells and B cells); Fugger et al. (1994) Expression of HLA-DR4 and human CD4 transgenes in mice determines the variable region β-chain T-cell repertoire and mediates an HLA-D-restricted immune response, Proc. Natl. Acad. Sci. USA, 91:6151-55 (CD4 expressed on all CD3+ thymocytes and B cells). Thus, in one embodiment, there may be a benefit in developing a genetically modified animal that retains endogenous mouse promoter and/or other regulatory elements in order for the animal to produce T cells that are capable of undergoing T cell development and lineage choice.

Thus, in various embodiments, the invention provides a genetically modified non-human animal, comprising, e.g., at its endogenous T cell co-receptor locus (e.g., CD4 locus), a nucleotide sequence encoding a chimeric human/non-human T cell co-receptor polypeptide. In one embodiment, a human portion of the chimeric polypeptide comprises all or substantially all of an extracellular portion (or part thereof, e.g., one or more extracellular domains, e.g., at least two consecutive extracellular domains) of a human T cell co-receptor. In one embodiment, a non-human portion of the chimeric polypeptide comprises transmembrane and cytoplasmic domains of a non-human T cell co-receptor. In one embodiment, the non-human animal expresses a functional chimeric T cell co-receptor polypeptide. Thus, in one aspect, the invention provides a genetically modified non-human animal comprising at its endogenous CD4 locus a nucleotide sequence encoding a chimeric human/non-human CD4 polypeptide, wherein a human portion of the chimeric polypeptide comprises all or substantially all of an extracellular portion of a human CD4, wherein a non-human portion comprises at least transmembrane and cytoplasmic domains of a non-human CD4, and wherein the animal expresses a functional chimeric CD4 polypeptide. In one aspect, the non-human animal only expresses the humanized CD4 polypeptide, i.e., chimeric human/non-human CD4 polypeptide, and does not express a functional endogenous non-human CD4 protein from its endogenous CD4 locus.

In one embodiment, the human portion of the chimeric human/non-human CD4 polypeptide comprises all or substantially all of the extracellular portion of a human CD4 polypeptide. In another embodiment, the human portion of the chimeric human/non-human CD4 polypeptide comprises at least all or substantially all of the MHC II binding domain of the human CD4 polypeptide (e.g., a substantial portion of human D1 and D2 domains); in one embodiment, the human portion of the chimeric human/non-human CD4 polypeptide comprises all or substantially all of D1, D2, and D3 domains of the human CD4 polypeptide; in yet another embodiment, the human portion of the chimeric human/non-human CD4 polypeptide comprises all or substantially all of immunoglobulin-like domains of CD4, e.g., domains termed D1, D2, D3, and D4. In yet another embodiment, the human portion of the chimeric human/non-human CD4 polypeptide comprises in its human portion all or substantially all of the human CD4 sequence that is responsible for interacting with MHC II and/or extracellular portion of a T cell receptor. In yet another embodiment, the human portion of the chimeric human/non-human CD4 polypeptide comprises all or substantially all of the extracellular portion of the human CD4 that is responsible for interacting with MHC II and/or the variable domain of a T cell receptor. Therefore, in one embodiment, the nucleotide sequence encoding the human portion of the chimeric CD4 polypeptide comprises all or substantially all of the coding sequence of domains D1-D2 of the human CD4 (e.g., a portion of exon 3 and exons 4-5 of the human CD4 gene); in another embodiment, it comprises all or substantially all of the coding sequence of D1-D3 of the human CD4 (e.g., portion of exon 3 and exons 4-6 of the human CD4). Thus, in one embodiment, the nucleotide sequence encoding chimeric human/non-human CD4 comprises nucleotide sequences encoding all or substantially all D1-D3 domains of the human CD4. In another embodiment, the nucleotide sequence encoding the human portion of the chimeric CD4 polypeptide comprises the coding sequence of D1-D4 domains of the human CD4 gene. In another embodiment, the nucleotide sequence may comprise the nucleotide sequence encoding mouse CD4 signal peptide, e.g., region encoded by portions of exons 2-3 of the mouse gene. In another embodiment, the nucleotide sequence may comprise the nucleotide sequence encoding a human CD4 signal peptide. In one embodiment, the chimeric human/non-human CD4 polypeptide comprises an amino acid sequence set forth in SEQ ID NO:78, and the human portion of the chimeric polypeptide spans about amino acids 27-319 of SEQ ID NO:78 (set forth separately in SEQ ID NO:79).

In one embodiment, the non-human animal expresses a chimeric human/non-human CD4 polypeptide sequence. In one embodiment, a human portion of the chimeric CD4 sequence comprises one or more conservative or non-conservative modifications.

In one aspect, a non-human animal that expresses a human CD4 sequence is provided, wherein the human CD4 sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human CD4 sequence. In a specific embodiment, the human CD4 sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the human CD4 sequence described in the Examples. In one embodiment, the human CD4 sequence comprises one or more conservative substitutions. In one embodiment, the human CD4 sequence comprises one or more non-conservative substitutions.

In some embodiments, a portion, e.g., a human portion of the chimeric CD4, may comprise substantially all of the sequence indicated herein (e.g., substantially all of a protein domain indicated herein). Substantially all sequence generally includes 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acids believed to represent a particular portion of the protein (e.g., a particular functional domain, etc.). One skilled in the art would understand that the boundaries of a functional domain may vary slightly depending on the alignment and domain prediction methods used.

In one aspect, the non-human portion of the chimeric human/non-human CD4 polypeptide comprises at least transmembrane and cytoplasmic domains of the non-human CD4 polypeptide. Due to the important functions served by CD4 cytoplasmic domain, retention of the endogenous non-human (e.g., mouse) sequence in genetically engineered animals ensures preservation of proper intracellular signaling and other functions of the co-receptor. In one embodiment, the non-human animal is a mouse, and the non-human CD4 polypeptide is a mouse CD4 polypeptide. Although a specific mouse CD4 sequence is described in the Examples, any suitable sequence derived therefrom, e.g., sequence comprising conservative/non-conservative amino acid substitutions, is encompassed herein. In one embodiment, the non-human portion of the chimeric CD4 co-receptor comprises any sequence of the endogenous CD4 that has not been humanized.

The non-human animal described herein may comprise at its endogenous locus a nucleotide sequence encoding a chimeric human/non-human CD4 polypeptide. In one aspect, this results in a replacement of a portion of an endogenous CD4 gene with a nucleotide sequence encoding a portion of a human CD4 polypeptide. In one embodiment, such replacement is a replacement of endogenous nucleotide sequence encoding, e.g., all or substantially all of the extracellular domain of a non-human CD4, e.g., a sequence encoding at least all or substantially all of the first immunoglobulin-like domain (i.e., D1) of a non-human CD4 (e.g., a sequence encoding all or substantially all of domains D1-D2 of a non-human CD4, e.g., a sequence encoding all or substantially all of domains D1-D3 of a non-human CD4, e.g., a sequence encoding all or substantially all of domains D1-D4 of a non-human CD4), with a human nucleotide sequence encoding the same. In one embodiment, the replacement results in a chimeric protein comprising human CD4 sequence that is responsible for interacting with MHC II and/or extracellular portion of a T cell receptor. In yet another embodiment, the replacement results in a chimeric protein comprising human CD4 sequence that is responsible for interacting with MHC II and/or variable domain of a T cell receptor. In one embodiment, the replacement does not comprise a replacement of a CD4 sequence encoding at least transmembrane and cytoplasmic domains of a non-human CD4 polypeptide. Thus, in one aspect, the non-human animal expresses a chimeric human/non-human CD4 polypeptide from the endogenous non-human CD4 locus. In yet another embodiment, the replacement results in a protein comprising a polypeptide sequence set forth in SEQ ID NO:78.

In one embodiment, the nucleotide sequence of the chimeric human/non-human CD4 locus (e.g., chimeric human/rodent CD4 locus, e.g., chimeric human/mouse CD4 locus) described herein is provided. In one aspect, because the chimeric human/non-human (e.g., human/rodent, e.g., human/mouse) CD4 sequence is placed at the endogenous non-human (e.g., rodent, e.g., mouse) CD4 locus, it retains the CD4 enhancer element located upstream of the first CD4 exon. In one embodiment, the replacement at the endogenous non-human (e.g., rodent, e.g., mouse) CD4 locus comprises a replacement of, e.g., a portion of exon 3 encoding D1, and exons 4-6 encoding the rest of D1 and D2-D3 of CD4 polypeptide; thus, in one aspect, the chimeric CD4 locus retains the cis-acting silencer located in intron 1 of the non-human (e.g., mouse) CD4 gene. Thus, in one embodiment, the chimeric locus retains endogenous non-human (e.g., rodent, e.g., mouse) CD4 promoter and regulatory elements. In another embodiment, the chimeric locus may contain human promoter and regulatory elements to the extent those allow proper CD4 expression, CD4+ T cell development, CD4 lineage choice, and co-receptor function. Thus, in some aspects, the animals of the invention comprise a genetic modification that does not alter proper lineage choice and development of T cells. In one aspect, the animals (e.g., rodents, e.g., mice) of the invention do not express chimeric CD4 polypeptide on immune cells other than cells that normally express CD4. In one aspect, animals do not express CD4 on B cells or mature CD8+ T cells. In one embodiment, the replacement results in retention of elements that allow proper spatial and temporal regulation of CD4 expression.

In various embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) that expresses a functional chimeric CD4 protein from a chimeric CD4 locus as described herein displays the chimeric protein on a cell surface, e.g., T cell surface. In one embodiment, the non-human animal expresses the chimeric CD4 protein on a cell surface in a cellular distribution that is the same as observed in a human. In one aspect, the CD4 protein of the invention is capable of interacting with an MHC II protein expressed on the surface of a second cell, e.g., an antigen presenting cell (APC).

Human or Humanized CD8

In various embodiments, the invention generally provides genetically modified non-human animals that comprise in their genome, e.g., at an endogenous CD8 locus, a nucleotide sequence encoding a human or humanized CD8 polypeptide; thus, the animals express a human or humanized CD8 polypeptide. In various embodiments, the invention provides non-human animals that comprise in their genome, e.g., at an endogenous CD8 locus, a nucleotide sequence encoding a human or humanized CD8α polypeptide and/or a nucleotide sequence encoding a human or humanized CD8β polypeptide. Thus, the genetically modified non-human animal of the invention expresses a human or humanized CD8α and/or a human or humanized CD8β polypeptide(s).

Human CD8 protein is typically expressed on cell surface as heterodimer of two polypeptides, CD8α and CD8β, although disulfide-linked homodimers and homomultimers have also been detected (e.g., in NK cells and intestinal γδ T cells, which express CD8αα). The genes encoding human CD8α and CD8β are located in close proximity to each other on chromosome 2. Nakayama et al. (1992) Recent Duplication of the Two Human CD8 β-chain genes, J. Immunol. 148:1919-27. CD8α protein contains a leader peptide, an immunoglobulin V-like region, a hinge region, a transmembrane domain and a cytoplasmic tail. Norment et al. (1989) Alternatively Spliced mRNA Encodes a Secreted Form of Human CD8α. Characterization of the Human CD8α gene, J. Immunol. 142:3312-19. The exons/introns of the CD8α gene are depicted schematically in FIG. 5B.

Human CD8β gene lies upstream of the CD8α gene on chromosome 2. Multiple isoforms generated by alternative splicing of CD8β gene have been reported, with one isoform predicted to lack a transmembrane domain and generate a secreted protein. Norment et al. (1988) A second subunit of CD8 is expressed in human T cells, EMBO J. 7:3433-39. The exons/introns of CD8β gene are also depicted schematically in FIG. 5B.

The membrane-bound CD8β protein contains an N-terminal signal sequence, followed by immunoglobulin V-like domain, a short extracellular hinge region, a transmembrane domain, and a cytoplasmic tail. See, Littman (1987) The structure of the CD4 and CD8 genes, Ann Rev. Immunol. 5:561-84. The hinge region is a site of extensive glycosylation, which is thought to maintain its conformation and protect the protein from cleavage by proteases. Leahy (1995) A structural view of CD4 and CD8, FASEB J. 9:17-25.

CD8 protein is commonly expressed on cytotoxic T cells, and interacts with MHC I molecules. The interaction is mediated through CD8 binding to the α₃ domain of MHC I. Although binding of MHC class I to CD8 is about 100-fold weaker than binding of TCR to MHC class I, CD8 binding enhances the affinity of TCR binding. Wooldridge et al. (2010) MHC Class I Molecules with Superenhanced CD8 Binding Properties Bypass the Requirement for Cognate TCR Recognition and Nonspecifically Activate CTLs, J. Immunol. 184:3357-3366.

CD8 binding to MHC class I molecules is species-specific; the mouse homolog of CD8, Lyt-2, was shown to bind H-2D^(d) molecules at the α3 domain, but it did not bind HLA-A molecules. Connolly et al. (1988) The Lyt-2 Molecule Recognizes Residues in the Class I α3 Domain in Allogeneic Cytotoxic T Cell Responses, J. Exp. Med. 168:325-341. Differential binding was presumably due to CDR-like determinants (CDR1- and CDR2-like) on CD8 that were not conserved between humans and mice. Sanders et al. (1991) Mutations in CD8 that Affect Interactions with HLA Class I and Monoclonal Anti-CD8 Antibodies, J. Exp. Med. 174:371-379; Vitiello et al. (1991) Analysis of the HLA-restricted Influenza-specific Cytotoxic T Lymphocyte Response in Transgenic Mice Carrying a Chimeric Human-Mouse Class I Major Histocompatibility Complex, J. Exp. Med. 173:1007-1015; and, Gao et al. (1997) Crystal structure of the complex between human CD8aa and HLA-A2, Nature 387:630-634. It has been reported that CD8 binds HLA-A2 in a conserved region of the α3 domain (at position 223-229). A single substitution (V245A) in HLA-A reduced binding of CD8 to HLA-A, with a concomitant large reduction in T cell-mediated lysis. Salter et al. (1989), Polymorphism in the α₃ domain of HLA-A molecules affects binding to CD8, Nature 338:345-348. In general, polymorphism in the α3 domain of HLA-A molecules also affected binding to CD8. Id. In mice, amino acid substitution at residue 227 in H-2D^(d) affected the binding of mouse Lyt-2 to H-2D^(d), and cells transfected with a mutant H-2D^(d) were not lysed by CD8+ T cells. Potter et al. (1989) Substitution at residue 227 of H-2 class I molecules abrogates recognition by CD8-dependent, but not CD8-independent, cytotoxic T lymphocytes, Nature 337:73-75. Thus, expression of human or humanized CD8 may be beneficial for studying T cell responses to antigen presented by human or humanized MHC I.

Similarly to CD4, the cytoplasmic domain of CD8 interacts with tyrosine kinase Lck, which in turn leads to T cell activation. Although Lck seems to interact with the cytoplasmic domain of CD8α, it appears that this interaction is regulated by the presence of the cytoplasmic domain of CD8β because mutations or deletion of CD8β cytoplasmic domain resulted in reduced CD8α-associated Lck activity. Irie et al. (1998) The cytoplasmic domain of CD8β Regulates Lck Kinase Activation and CD8 T cell Development, J. Immunol. 161:183-91. The reduction in Lck activity was associated with impairment in T cell development. Id.

Expression of CD8 on appropriate cells, e.g., cytotoxic T cells, is tightly regulated by a variety of enhancer elements located throughout the CD8 locus. For instance, at least 4 regions of DNAse I-hypersensitivity, regions often associated with regulator binding, have been identified at the CD8 locus. Hosert et al. (1997) A CD8 genomic fragment that directs subset-specific expression of CD8 in transgenic mice, J. Immunol. 158:4270-81. Since the discovery of these DNAse I-hypersensitive regions at CD8 locus, at least 5 enhancer elements have been identified, spread throughout the CD8 locus, that regulate expression of CD8α and/or β in T cells of various lineages, including DP, CD8 SP T cells, or cells expressing γδTCR. See, e.g., Kioussis et al. (2002) Chromatin and CD4, CD8A, and CD8B gene expression during thymic differentiation, Nature Rev. 2:909-919 and Online Erratum; Ellmeier et al. (1998) Multiple Development Stage-Specific Enhancers Regulate CD8 Expression in Developing Thymocytes and in Thymus-Independent T cells, Immunity 9:485-96.

Thus, similarly to the benefit derived from retaining endogenous CD4 promoter and regulatory elements for human or humanized CD4 genetically modified animals, in some embodiments, there may be a benefit in developing a genetically modified non-human animal that retains endogenous mouse promoter and regulatory elements that would control expression of human or humanized CD8. There may be a particular benefit in creating genetically modified animals comprising a replacement of endogenous non-human sequences encoding CD8α and/or β proteins with those encoding human or humanized CD8α and/or β proteins, as described herein.

In various embodiments, the invention provides a genetically modified non-human animal comprising in its genome, e.g., at its endogenous CD8 locus, at least one nucleotide sequence encoding a chimeric human/non-human CD8 polypeptide (e.g., CD8α and/or β polypeptide), wherein a human portion of the polypeptide comprises all or substantially all of an extracellular portion (or a part thereof, e.g., an extracellular domain) of a human CD8 polypeptide (e.g., CD8α and/or β), wherein a non-human portion comprises at least transmembrane and cytoplasmic domains of a non-human CD8 (e.g., CD8α and/or β), and wherein the animal expresses the chimeric CD8 polypeptide (e.g., CD8α and/or β polypeptide). Thus, in one embodiment, the invention provides a genetically modified non-human animal comprising at its endogenous non-human CD8 locus a first nucleotide sequence encoding a chimeric human/non-human CD8α polypeptide and a second nucleotide sequence encoding a chimeric human/non-human CD8β polypeptide, wherein the first nucleotide sequence comprises a sequence that encodes all or substantially all of the extracellular portion of a human CD8α polypeptide and at least transmembrane and cytoplasmic domains of a non-human CD8α polypeptide, and wherein the second nucleotide sequence comprises a sequence that encodes all or substantially all of the extracellular portion of a human CD8β polypeptide and at least transmembrane and cytoplasmic domains of a non-human CDβ polypeptide, wherein the animal expresses a functional chimeric human/non-human CD8 protein. In one aspect, the non-human animal only expresses a humanized CD8 polypeptide (e.g., chimeric human/non-human CD8α and/or β polypeptide), and does not express a corresponding functional non-human CD8 polypeptide(s) from the endogenous CD8 locus.

In one embodiment, the chimeric human/non-human CD8α polypeptide comprises in its human portion all or substantially all of the extracellular portion of a human CD8α polypeptide. In one embodiment, the human portion of the chimeric CD8α polypeptide comprises at least the MHC I binding domain of the human CD8α polypeptide. In one embodiment, the human portion of the chimeric CD8α polypeptide comprises the sequence of at least all or substantially all of the immunoglobulin V-like domain of the human CD8α. In one embodiment, the nucleotide sequence encoding the human portion of the chimeric CD8α polypeptide comprises at least the exons that encode an extracellular portion of the human CD8α polypeptide. In one embodiment, the nucleotide sequence comprises at least the exons that encode the Ig V-like domains. In one embodiment, the extracellular portion of a human CD8α polypeptide is a region encompassing the portion of the polypeptide that is not transmembrane or cytoplasmic domain. In one embodiment, the nucleotide sequence encoding the chimeric human/non-human CD8α polypeptide comprises the sequence encoding a non-human (e.g., rodent, e.g., mouse) CD8α signal peptide. Alternatively, the nucleotide sequence may comprise the sequence encoding a human CD8α signal sequence. In one embodiment, the chimeric human/non-human CD8α polypeptide comprises an amino acid sequence set forth in SEQ ID NO:88, and the human portion of the chimeric polypeptide is set forth at amino acids 28-179 of SEQ ID NO:88 (represented separately in SEQ ID NO:89).

Similarly, in one embodiment, the chimeric human/non-human CD8β polypeptide comprises in its human portion all or substantially all of the extracellular portion of a human CD8β polypeptide. In one embodiment, the human portion of the chimeric CD8β polypeptide comprises the sequence of all or substantially all of the immunoglobulin V-like domain of human CD8β. In one embodiment, the nucleotide sequence encoding the human portion of the chimeric CD8β polypeptide comprises at least the exons that encode the extracellular portion of the human CD8β polypeptide. In one embodiment, the nucleotide sequence encoding the human portion of the chimeric human/non-human CD8β polypeptide comprises at least the exons that encode the IgG V-like domain of human CD8β. In one embodiment, the nucleotide sequence encoding the chimeric human/non-human CD8β polypeptide comprises the sequence encoding a non-human (e.g., rodent, e.g., mouse) CD8β signal peptide. Alternatively, the nucleotide sequence may comprise the sequence encoding a human CD8β signal sequence. In one embodiment, the chimeric human/non-human CD8β polypeptide comprises an amino acid sequence set forth in SEQ ID NO:83, and the human portion of the chimeric polypeptide is set forth at amino acids 15-165 of SEQ ID NO:83 (represented separately in SEQ ID NO:84).

In one embodiment, the non-human animal expresses a chimeric human/non-human CD8α and/or CD8β polypeptides. In some embodiments, the human portion of the chimeric human/non-human CD8α and/or β polypeptide comprises one or more conservative or nonconservative modification(s).

In one aspect, a non-human animal that expresses a human CD8α and/or β polypeptide sequence is provided, wherein the human CD8α and/or β polypeptide sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human CD8α and/or β polypeptide sequence, respectively. In a specific embodiment, the human CD8α and/or β polypeptide sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the respective human CD8α and/or β polypeptide sequence described in the Examples. In one embodiment, the human CD8α and/or β polypeptide sequence comprises one or more conservative substitutions. In one embodiment, the human CD8α and/or β polypeptide sequence comprises one or more non-conservative substitutions.

In some embodiments, a portion, e.g., a human portion of the chimeric CD8, may comprise substantially all of the sequence indicated herein (e.g., substantially all of a protein domain indicated herein). Substantially all sequence generally includes 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acids believed to represent a particular portion of the protein (e.g., a particular functional domain, etc.). One skilled in the art would understand that the boundaries of a functional domain may vary slightly depending on the alignment and domain prediction methods used.

In one aspect, the non-human portion of the chimeric human/non-human CD8α and/or β polypeptide comprises at least transmembrane and/or cytoplasmic domain of the non-human CD8α and/or β polypeptide, respectively. Due to the important functions served by CD8 cytoplasmic domain, retention of the endogenous non-human (e.g., mouse) sequence in genetically engineered animals ensures preservation of proper intracellular signaling and other functions of the co-receptor. In one embodiment, the non-human animal is a mouse, and the non-human CD8α and/or β polypeptide is a mouse CD8α and/or β polypeptide, respectively. Although specific mouse CD8α and β sequences are described in the Examples, any suitable sequence derived therefrom, e.g., sequence comprising conservative/non-conservative amino acid substitutions, is encompassed herein. In one embodiment, the non-human animal (e.g., rodent, e.g., mouse) retains any endogenous sequence that has not been humanized.

The non-human animal described herein may comprise at its endogenous locus a nucleotide sequence encoding a chimeric human/non-human CD8α and/or β polypeptide. In one aspect, this results in a replacement of a portion of an endogenous CD8α gene with a nucleotide sequence encoding a portion of a human CD8α polypeptide, and/or a replacement of a portion of an endogenous CD8β gene with a nucleotide sequence encoding a portion of a human CD8β polypeptide. In one embodiment, such replacement is a replacement of endogenous nucleotide sequence encoding all or substantially all of extracellular portion of a non-human CD8α and/or β with a human nucleotide with a human nucleotide sequence encoding the same. In one embodiment, such replacement is a replacement of a sequence encoding at least all or substantially all of the immunoglobulin V-like domain of a non-human CD8α and/or β with a human nucleotide sequence encoding the same. In one embodiment, the replacement does not comprise a replacement of a CD8α and/or β sequence encoding transmembrane and cytoplasmic domain of a non-human CD8α and/or β polypeptide. Thus, the non-human animal expresses a chimeric human/non-human CD8α and/or β polypeptide from the endogenous non-human CD8 locus. In yet another embodiment, the replacement results in a CD8α and/or β protein comprising a polypeptide sequence set forth in SEQ ID NO:88 and/or 84, respectively.

In one embodiment, the nucleotide sequence of the chimeric human/non-human CD8 locus (e.g., chimeric rodent CD8 locus, e.g., chimeric mouse CD8 locus) is provided. In one aspect, because the chimeric human/non-human (e.g., human/rodent, e.g., human/mouse) CD8α and/or β sequence is placed at respective endogenous non-human (e.g., rodent, e.g., mouse) CD8α and/or β locus, it retains endogenous CD8α and/or β promoter and regulatory elements. In another embodiment, the chimeric locus may contain human CD8α and/or β promoter and regulatory elements to the extent those allow proper CD8α and/or β expression (proper spatial and temporal protein expression), CD8+ T cell development, CD8 lineage choice, and co-receptor function. Thus, in one aspect, the animals of the invention comprise a genetic modification that does not alter proper lineage choice and development of T cells. In one aspect, the animals (e.g., rodents, e.g., mice) of the invention do not express chimeric CD8 protein on immune cells other than cells that normally express CD8, e.g., animals do not express CD8 on B cells or mature CD4+ T cells. In one embodiment, the replacement results in retention of elements that allow proper spatial and temporal regulation of CD8α and/or β expression.

In various embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) that expresses a functional chimeric CD8 protein (e.g., CD8αβ or CD8αα) from a chimeric CD8 locus as described herein displays the chimeric protein on a cell surface. In one embodiment, the non-human animal expresses the chimeric CD8 protein on a cell surface in a cellular distribution that is the same as observed in a human. In one aspect, the CD8 protein of the invention is capable of interacting with an MHC I protein expressed on the surface of a second cell.

Human or Humanized T Cell Receptor

Disclosed herein are genetically modified non-human animals comprising a substantially humanized T cell immune system. In some embodiment a non-human animal as disclosed herein comprises, e.g., in its genome, (a) a nucleotide sequence encoding a chimeric human/non-human T cell co-receptor, wherein the human portion of the chimeric T cell co-receptor polypeptide is encoded by a sequence encoding an extracellular domain of a human T cell co-receptor, and wherein the sequence encoding the extracellular domain of a human T cell co-receptor is operably linked to a nucleotide comprising a sequence encoding a non-human T cell co-receptor transmembrane and/or cytoplasmic domain; (b) an unrearranged T cell receptor (TCR) variable gene region comprising at least one human V segment, optionally at least on human D segment, and at least one human J segment, wherein the unrearranged V, optionally D, and J segments of the TCR variable region gene can recombine to form a rearranged gene operably linked to a non-human TCR constant gene sequence; and (c) a nucleic acid sequence encoding a chimeric human/non-human MHC polypeptide, wherein a human portion of the chimeric MHC polypeptide comprises an extracellular domain of a human MHC polypeptide that associates with the human portion of the chimeric T cell co-receptor polypeptide. Optionally, the non-human animal also comprises a human or humanized β2 microglobulin polypeptide.

Accordingly, in various embodiments, the invention generally provides genetically modified non-human animals wherein the non-human animals comprise in the genome unrearranged humanized TCR variable gene loci, e.g., an unrearranged human TCR variable gene region comprising human TCR variable segments capable of recombining to form a rearranged TCR variable gene sequence. TCR locus or TCR gene locus (e.g., TCRα locus or TCRβ locus), as used herein, refer to the genomic DNA comprising the TCR coding region, including the entire TCR coding region, including unrearranged V(D)J sequences, enhancer sequence, constant sequence(s), and any upstream or downstream (UTR, regulatory regions, etc.), or intervening DNA sequence (introns, etc.). TCR variable locus, TCR variable region, or TCR variable gene locus (e.g., TCRα variable gene locus or TCRβ variable gene locus), refers to genomic DNA that includes TCR variable region segments (V(D)J region) but excludes TCR constant sequences and, in various embodiments, enhancer sequences. Other sequences may be included in the TCR variable gene locus for the purposes of genetic manipulation (e.g., selection cassettes, restriction sites, etc.), and these are encompassed herein.

T cells bind epitopes on small antigenic determinants on the surface of antigen-presenting cells that are associated with a major histocompatibility complex (MHC; in mice) or human leukocyte antigen (HLA; in humans) complex. T cells bind these epitopes through a T cell receptor (TCR) complex on the surface of the T cell. T cell receptors are heterodimeric structures composed of two types of chains: an α (alpha) and β (beta) chain, or a γ (gamma) and δ (delta) chain. The a chain is encoded by the nucleic acid sequence located within the α locus (on human or mouse chromosome 14), which also encompasses the entire δ locus, and the β chain is encoded by the nucleic acid sequence located within the β locus (on mouse chromosome 6 or human chromosome 7). The majority of T cells has an αβ TCR; while a minority of T cells bears a γδ TCR. Interactions of TCRs with MHC class I (presenting to CD8+ T cells) and MHC class II (presenting to CD4+ T cells) molecules are shown in FIG. 1 (closed symbols represent non-human sequences; striped symbols represent human sequences, showing one particular embodiment of the TCR protein of the present invention).

T cell receptor a and β polypeptides (and similarly γ and δ polypeptides) are linked to each other via a disulfide bond. Each of the two polypeptides that make up the TCR contains an extracellular domain comprising constant and variable regions, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and the cytoplasmic tail also being a part of the constant region). The variable region of the TCR determines its antigen specificity, and similar to immunoglobulins, comprises three complementary determining regions (CDRs). Also similar to immunoglobulin genes, T cell receptor variable gene loci (e.g., TCRα and TCRβ loci) contain a number of unrearranged V(D)J segments (variable (V), joining (J), and in TCRβ and δ, diversity (D) segments). During T cell development in the thymus, TCRα variable gene locus undergoes rearrangement, such that the resultant TCR α chain is encoded by a specific combination of VJ segments (Vα/Jα sequence); and TCRβ variable gene locus undergoes rearrangement, such that the resultant TCR β chain is encoded by a specific combination of VDJ segments (Vβ/Dβ/Jβ sequence).

Interactions with thymic stroma trigger thymocytes to undergo several developmental stages, characterized by expression of various cell surface markers. A summary of characteristic cell surface markers at various developmental stages in the thymus is presented in Table 1. Rearrangement at the TCRβ variable gene locus begins at the DN2 stage and ends during the DN4 stage, while rearrangement of the TCRα variable gene locus occurs at the DP stage. After the completion of TCRβ locus rearrangement, the cells express TCRβ chain at the cell surface together with the surrogate α chain, pTα. See, Janeway's Immunobiology, Chapter 7, 7^(th) Ed., Murphy et al. eds., Garland Science, 2008.

TABLE 1 Developmental Stages of T cells in the Thymus Developmental Stage DN1 DN2 DN3 DN4 DP SP Marker(s) CD44+/CD25− CD44+/0D25+ CD44^(low)/CD25+ CD44−/CD25− CD4+/CD8+ CD4+ or CD8+

Naive CD4+ and CD8+ T cells exit the thymus and enter the peripheral lymphoid organs (e.g., spleen) where they are exposed to antigens and are activated to clonally expand and differentiate into a number of effector T cells (Teff), e.g., cytotoxic T cells, T_(REG) cells, T_(H)17 cells, T_(H)1 cells, T_(H)2 cells, etc. Subsequent to infection, a number of T cells persist as memory T cells, and are classified as either central memory T cells (Tcm) or effector memory T cells (Tem). Sallusto et al. (1999) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions, Nature 401:708-12 and Commentary by Mackay (1999) Dual personality of memory T cells, Nature 401:659-60. Sallusto and colleagues proposed that, after initial infection, Tem cells represent a readily available pool of antigen-primed memory T cells in the peripheral tissues with effector functions, while Tcm cells represent antigen-primed memory T cells in the peripheral lymphoid organs that upon secondary challenge can become new effector T cells. While all memory T cells express CD45RO isoform of CD45 (naïve T cells express CD45RA isoform), Tcm are characterized by expression of L-selectin (also known as CD62L) and CCR7+, which are important for binding to and signaling in the peripheral lymphoid organs and lymph nodes. Id. Thus, all T cells found in the peripheral lymphoid organs (e.g., naïve T cells, Tcm cells, etc.) express CD62L. In addition to CD45RO, all memory T cells are known to express a number of different cell surface markers, e.g., CD44. For summary of various cell surface markers on T cells, see Janeway's Immunobiology, Chapter 10, supra.

While TCR variable domain functions primarily in antigen recognition, the extracellular portion of the constant domain, as well as transmembrane, and cytoplasmic domains of the TCR also serve important functions. A complete TCR receptor complex requires more than the a and β or γ and δ polypeptides; additional molecules required include CD3γ, CD3δ, and CD3ε, as well as the ζ chain homodimer (CC). At the completion of TCRβ rearrangement, when the cells express TCRβ/pTα, this pre-TCR complex exists together with CD3 on the cell surface. TCRα (or pTα) on the cell surface has two basic residues in its transmembrane domain, one of which recruits a CD3γε heterodimer, and another recruits ζζ via their respective acidic residues. TCRβ has an additional basic residue in its transmembrane domain that is believed to recruit CD3δε heterodimer. See, e.g., Kuhns et al. (2006) Deconstructing the Form and Function of the TCR/CD3 Complex, Immunity 24:133-39; Wucherpfennig et al. (2009) Structural Biology of the T-cell Receptor: Insights into Receptor Assembly, Ligand Recognition, and Initiation of Signaling, Cold Spring Harb. Perspect. Biol. 2:a005140. The assembled complex, comprising TCRαβ heterodimer, CD3γε, CD3δε, and ζζ, is expressed on the T cell surface. The polar residues in the transmembrane domain have been suggested to serve as quality control for exiting endoplasmic reticulum; it has been demonstrated that in the absence of CD3 subunits, TCR chains are retained in the ER and targeted for degradation. See, e.g., Call and Wucherpfennig (2005) The T Cell Receptor: Critical Role of the Membrane Environment in Receptor Assembly and Function, Annu. Rev. Immunol. 23:101-25.

CD3 and ζ chains of the assembled complex provide components for TCR signaling as TCRαβ heterodimer (or TCRγδ heterodimer) by itself lacks signal transducing activity. The CD3 chains possess one Immune-Receptor-Tyrosine-based-Activation-Motif (ITAM) each, while the ζ chain contains three tandem ITAMs. ITAMs contain tyrosine residues capable of being phosphorylated by associated kinases. Thus, the assembled TCR-CD3 complex contains 10 ITAM motifs. See, e.g., Love and Hayes (2010) ITAM-Mediated Signaling by the T-Cell Antigen Receptor, Cold Spring Harb. Perspect. Biol. 2:e002485. Following TCR engagement, ITAM motifs are phosphorylated by Src family tyrosine kinases, Lck and Fyn, which initiates a signaling cascade, resulting in Ras activation, calcium mobilization, actin cytoskeleton rearrangements, and activation of transcription factors, all ultimately leading to T cell differentiation, proliferation, and effector actions. Id., see also, Janeway's Immunobiology, supra; both incorporated herein by reference.

Additionally, TCRβ transmembrane and cytoplasmic domains are thought to have a role in mitochondrial targeting and induction of apoptosis; in fact, naturally occurring N-terminally truncated TCRβ molecules exist in thymocytes. Shani et al. (2009) Incomplete T-cell receptor-β peptides target the mitochondrion and induce apoptosis, Blood 113:3530-41. Thus, several important functions are served by the TCR constant region (which, in various embodiments, comprises a portion of extracellular as well as transmembrane and cytoplasmic domains); and in various embodiments the structure of this region should be taken into consideration when designing humanized TCRs or genetically modified non-human animals expressing the same.

Mice transgenic for rearranged T cell receptor sequences are known in the art. The present invention relates to genetically modified non-human animals (e.g., rodents, e.g., rats, mice) that comprise unrearranged human or humanized T cell variable gene loci that are capable of rearranging to form nucleic acid sequences that encode human T cell receptor variable domains, including animals that comprise T cells that comprise rearranged human variable domains and non-human (e.g., mouse or rat) constant regions. The present invention also provides non-human animals (e.g., rodents, e.g., rats, mice) that are capable of generating a diverse repertoire of human T cell receptor variable region sequences; thus, the present invention provides non-human animals that express TCRs with fully human variable domains in response to an antigen of interest and that bind an epitope of the antigen of interest. In some embodiments, provided are non-human animals that generate a diverse T cell receptor repertoire capable of reacting with various antigens, including but not limited to antigens presented by APCs.

In one embodiment, the invention provides genetically modified non-human animals (e.g., rodents, e.g., rats, mice) that comprise in their genome unrearranged human TCR variable region segments (V(D)J segments), wherein the unrearranged human TCR variable region segments replace, at an endogenous non-human (e.g., rodent) TCR variable gene locus (e.g., TCRα, β, δ, and/or γ variable gene locus), endogenous non-human TCR variable region segments. In one embodiment, unrearranged human TCR variable gene locus replaces endogenous non-human TCR variable gene locus.

In another embodiment, the invention provides genetically modified non-human animals (e.g., rodents, e.g., rats, mice) that comprise in their genome unrearranged human TCR variable region segments (V(D)J segments), wherein the unrearranged human TCR variable region segments are operably linked to a non-human TCR constant region gene sequence resulting in a humanized TCR locus, wherein the humanized TCR locus is at a site in the genome other than the endogenous non-human TCR locus. Thus, in one embodiment, a non-human animal (e.g., rodent, e.g., mouse, rat) comprising a transgene that comprises unrearranged human TCR variable region segments operably linked to non-human TCR constant region gene sequence is also provided.

In one aspect, the genetically modified non-human animals of the invention comprise in their genome human TCR variable region segments, while retaining non-human (e.g., rodent, e.g., mouse, rat) TCR constant gene sequence(s) that encode TCR constant domains. In various embodiments, a TCR constant domain includes the transmembrane domain and the cytoplasmic tail of the TCR. Thus, in various embodiments of the present invention, the genetically modified non-human animals retain endogenous non-human TCR transmembrane domain and cytoplasmic tail. In other embodiments, non-human animals comprise non-human non-endogenous TCR constant gene sequences, e.g., encoding non-human non-endogenous TCR transmembrane domain and cytoplasmic tail. As indicated above, the constant domain of the TCR participates in a signaling cascade initiated during antigen-primed T cell activation; thus, endogenous TCR constant domain interacts with a variety of non-human anchor and signaling proteins in the T cell. Thus, in one aspect, the genetically modified non-human animals of the invention express humanized T cell receptors that retain the ability to recruit a variety of endogenous non-human anchor or signaling molecules, e.g., CD3 molecules (e.g., CD3γ, CD3δ, CD3ε), the ζ chain, Lck, Fyn, ZAP-70, etc. A nonlimiting list of molecules that are recruited to the TCR complex is described in Janeway's Immunobiology, supra. It is believed that the ability of T cell development and T cell differentiation processes in the non-human animals to proceed and allow for a robust immune response may be due, at least in part, to the placement of variable regions at the endogenous mouse loci and the maintenance of mouse constant domains.

In some embodiments, a non-human animal is provided that comprises in its genome unrearranged human TCRα variable region segments, wherein the unrearranged human TCRα variable region segments are operably linked to a non-human TCRα constant region gene sequence resulting in a humanized TCRα locus. In one embodiment, the humanized TCRα locus is at a site in the genome other than the endogenous non-human TCRα locus. In another embodiment, the unrearranged human TCRα variable region segments replace endogenous non-human TCRα variable region segments while retaining endogenous non-human TCRα constant region gene sequence(s). In one embodiment, the unrearranged human TCRα variable gene locus replaces endogenous non-human TCRα variable gene locus. In some embodiments, replacement of an endogenous non-human TCRα variable region gene locus with the unrearranged human TCRα variable gene locus comprises a deletion or inactivation of a TCRδ variable gene locus. In other embodiments, replacement of an endogenous non-human TCRα variable region gene with the unrearranged human TCRα gene locus comprises a replacement of an endogenous TCRδ variable gene locus with unrearranged human TCRδ variable region segments. In some embodiments, the animal retains endogenous non-human TCRβ variable region and constant region gene sequence(s). Thus, the animal expresses a TCR that comprises a chimeric human/non-human (i.e., humanized) TCRα chain and a non-human TCRβ chain.

In some embodiments, a non-human animal is provided that comprises in its genome unrearranged human TCRδ variable region segments, wherein the unrearranged human TCRδ variable region segments are operably linked to a non-human TCRδ constant region gene sequence resulting in a humanized TCRδ locus. In one embodiment, the humanized TCRδ locus is at a site in the genome other than the endogenous non-human TCRδ locus. In another embodiment, the unrearranged human TCRδ variable region segments replace endogenous non-human TCRδ variable region segments while retaining endogenous non-human TCRδ constant region gene sequence(s). In one embodiment, the unrearranged human TCRδ variable gene locus replaces endogenous non-human TCRδ variable gene locus.

In other embodiments, a non-human animal is provided that comprises in its genome unrearranged human TCRβ variable region segments, wherein the unrearranged human TCRβ variable region segments are operably linked to a non-human TCRβ constant region gene sequence resulting in a humanized TCRβ locus. In one embodiment, the humanized TCRβ locus is at a site in the genome other than the endogenous non-human TCRβ locus. In another embodiment, the unrearranged human TCRβ variable region segments replace endogenous non-human TCRβ variable region segments while retaining endogenous non-human TCRβ constant region gene sequence(s). In one embodiment, the unrearranged human TCRβ variable gene locus replaces endogenous non-human TCRβ variable gene locus. In some embodiments, the animal retains endogenous non-human TCRα variable region and constant region gene sequence(s). Thus, the animal expresses a TCR that comprises a chimeric human/non-human (i.e., humanized) TCRβ chain and a non-human TCRα chain.

In some specific embodiments, the invention provides a genetically modified non-human animal (e.g., rodent, e.g., mouse or rat) that comprises in its genome (a) an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCRα constant gene sequence(s), (b) an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCRβ constant region gene sequence(s) and/or (c) an unrearranged TCRδ variable gene locus comprising at least one human Vδ segment, at least one human Dδ segment, and at least one human Jδ segment, operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCRδ constant region gene sequence. In some embodiments, a non-human animal as described herein comprises in its genome (a) an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCRα constant gene sequence(s), (b) an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCRβ constant gene sequence(s), (c) an unrearranged TCRδ variable gene locus comprising at least one human Vδ segment, at least one human Dδ segment, and at least one human Jδ segment, operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCRδ constant region gene sequence(s) and/or (d) an unrearranged TCRγ variable gene locus comprising at least one human Vγ segment, and at least one human Jγ segment, operably linked to an endogenous non-human (e.g., rodent, e.g., mouse or rat) TCRγ constant region gene sequence.

In various embodiments of the invention, the unrearranged human or humanized TCR variable gene locus (e.g., TCRα TCRβ and/or TCRδ variable gene locus) is comprised in the germline of the non-human animal (e.g., rodent, e.g., mouse or rat). In various embodiments, the replacements of TCR V(D)J segments by unrearranged human TCR V(D)J segments (e.g., Vα and Jα; Vβ and Dβ and Jβ; Vδ and Dδ and Jδ; Vγ and Jγ segments) are at an endogenous non-human TCR variable locus (or loci), wherein the unrearranged human V and J and/or V and D and J segments are operably linked to non-human TCR constant region gene sequences.

In some embodiments of the invention, the non-human animal comprises two copies of the unrearranged human or humanized TCRα variable gene locus, two copies of the unrearranged human or humanized TCRβ variable gene locus and/or two copies of the unrearranged human or humanized TCRδ variable gene locus. Thus, the non-human animal is homozygous for one or more unrearranged human or humanized TCRα, TCRβ and/or TCRδ variable gene loci. In some embodiments of the invention, the non-human animal comprises one copy of the unrearranged human or humanized TCRα variable gene locus, one copy of the unrearranged human or humanized TCRβ variable gene locus and/or one copy of the unrearranged human or humanized TCRδ variable gene locus. Thus, the non-human animal is heterozygous for unrearranged human or humanized TCRα, TCRβ and/or TCRδ variable gene locus. In other embodiment, a non-human animal is heterozygous or homozygous for unrearranged human or humanized TCRγ variable gene locus.

In one embodiment, the unrearranged TCRα variable gene locus comprising human variable region segments (e.g., human Vα and Jα segments) is positioned in the non-human genome such that the human variable region segments replace corresponding non-human variable region segments. In one embodiment, the unrearranged TCRα variable gene locus comprising human variable region segments replaces endogenous TCRα variable gene locus. In one aspect, endogenous non-human Vα and Jα segments are incapable of rearranging to form a rearranged Vα/Jα sequence. Thus, in one aspect, the human Vα and Jα segments in the unrearranged TCRα variable gene locus are capable of rearranging to form a rearranged human Vα/Jα sequence.

Similarly, in one embodiment, the unrearranged TCRβ variable gene locus comprising human variable region segments (e.g., human Vβ, Dβ, and Jβ segments) is positioned in the non-human genome such that the human variable region segments replace corresponding non-human variable region segments. In one embodiment, the unrearranged TCRβ variable gene locus comprising human variable region segments replaces endogenous TCRβ variable gene locus. In one aspect, endogenous non-human Vβ, Dβ, and Jβ segments are incapable of rearranging to form a rearranged Vβ/Dβ/Jβ sequence. Thus, in one aspect, the human Vβ, Dβ, and Jβ segments in the unrearranged TCRβ variable gene locus are capable of rearranging to form a rearranged human Vβ/Dβ/Jβ sequence.

In one embodiment, the unrearranged TCRδ variable gene locus comprising human variable region segments (e.g., human Vδ, Dδ, and Jδ segments) is positioned in the non-human genome such that the human variable region segments replace corresponding non-human variable region segments. In one embodiment, the unrearranged TCRδ variable gene locus comprising human variable region segments replaces endogenous TCRδ variable gene locus. In one aspect, endogenous non-human Vδ, Dδ, and Jδ segments are incapable of rearranging to form a rearranged Vδ/Dδ/Jδ sequence. Thus, in one aspect, the human Vδ, Dδ, and Jδ segments in the unrearranged TCRδ variable gene locus are capable of rearranging to form a rearranged human Vδ/Dδ/Jδ sequence.

In one embodiment, the unrearranged TCRγ variable gene locus comprising human variable region segments (e.g., human Vγ and Jγ segments) is positioned in the non-human genome such that the human variable region segments replace corresponding non-human variable region segments. In one embodiment, the unrearranged TCRγ variable gene locus comprising human variable region segments replaces endogenous TCRγ variable gene locus. In one aspect, endogenous non-human Vα and Jα segments are incapable of rearranging to form a rearranged Vγ/Jγ sequence. Thus, in one aspect, the human Vγ and Jγ segments in the unrearranged TCRγ variable gene locus are capable of rearranging to form a rearranged human Vγ/Jγ sequence.

In yet another embodiment, the unrearranged TCRα, β, β and/or γ variable gene loci comprising human variable region segments replace respective endogenous TCRα, β, δ, and γ variable gene loci. In one aspect, endogenous non-human Vα and Jα segments are incapable of rearranging to form a rearranged Vα/Jα sequence, endogenous non-human Vβ, Dβ, and Jβ segments are incapable of rearranging to form a rearranged Vβ/Dβ/Jβ sequence, endogenous Vδ, Dδ, and Jδ segments are incapable of rearranging to form a rearranged Vδ/Dδ/Jδ sequence and/or endogenous non-human Vγ and Jγ segments are incapable of rearranging to form a rearranged Vγ/Jγ sequence. Thus, in one aspect, the human Vα and Jα segments in the unrearranged TCRα variable gene locus are capable of rearranging to form a rearranged human Vα/Jα sequence, the human Vβ, Dβ, and Jβ segments in the unrearranged TCRβ variable gene locus are capable of rearranging to form a rearranged human Vβ/Dβ/Jβ sequence, the human Vδ, Dδ, and Jδ segments in the unrearranged TCRδ variable gene locus are capable of rearranged to form a rearranged human Vδ/Dδ/Jδ sequence and/or the human Vγ and Jγ segments in the unrearranged TCRγ variable gene locus are capable of rearranging to form a rearranged human Vγ/Jγ sequence.

In some aspects of the invention, the non-human animal comprising a humanized TCRα, TCRβ and/or TCR δ gene locus (comprising an unrearranged human TCRα, TCRβ and/or TCR δ variable gene locus) retains an endogenous non-human TCRα TCRβ and/or TCRδ variable gene locus. In one embodiment, the endogenous non-human TCRα, TCRβ and/or TCRδ variable gene locus is a non-functional locus. In one embodiment, the non-functional locus is an inactivated locus, e.g., an inverted locus (e.g., the coding nucleic acid sequence of the variable gene locus is in inverted orientation with respect to the constant region sequence, such that no successful rearrangements are possible utilizing variable region segments from the inverted locus). In one embodiment, the humanized TCRα, TCRβ and/or TCR δ variable gene locus is positioned between the endogenous non-human TCRα, TCRβ and/or TCRδ variable gene locus and the endogenous non-human TCRα, TCRβ and/or TCRδ constant gene locus, respectively. Similar chromosomal arrangements may be made for placing human or humanized TCRγ into the genome of a non-human animal, e.g., at a TCRγ locus.

The number, nomenclature, position, as well as other aspects of V and J and/or V, D, and J segments of the human and mouse TCR loci may be ascertained using the IMGT database, available at the website of the International Immunogenetics Information System (IMGT). The mouse TCRα variable locus is approximately 1.5 megabases and comprises a total of 110Vα and 60 Jα segments. The human TCRα variable locus is approximately 1 megabase and comprises a total of 54Vα and 61Jα segments, with 45Vα and 50Jα believed to be functional. Unless stated otherwise, the numbers of human V(D)J segments referred to throughout the specification refers to the total number of V(D)J segments. In one embodiment of the invention, the genetically modified non-human animal (e.g., rodent, e.g., mouse or rat) comprises at least one human Vα and at least one human Jα segment. In one embodiment, the non-human animal comprises a humanized TCRα locus that comprises 1, 2, 3, 4, 5, 6, 7, δ, 9, 10, 15, 20, 23, 25, 30, 35, 40, 45, 48, 50, or up to 54 human Vα segments. In some embodiments, the humanized TCRα locus comprises 2, δ, 23, 35, 48, or 54 human Vα segments. Thus, in some embodiments, the humanized TCRα locus in the non-human animal may comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 99%, or 100% of human Vα; in some embodiments, it may comprise about 2%, about 3%, about 15%, about 65%, about 90%, or 100% of human Vα.

In one embodiment, the non-human animal comprises a humanized TCRα locus that comprises a DNA fragment comprising a contiguous human sequence of human Vα40 to Vα41 (Vα segment is also referred to as “TRAV” or “TCRAV”) and a DNA fragment comprising a contiguous human sequence of 61 human Jα segments (Jα segment is also referred to as “TRAJ” or “TCRAJ”). A TCRA non-coding sequence refers to a contiguous non-coding sequence comprising non-coding recombinant signal sequences (RSSs) and other non-coding intergenic sequences found between any two consecutive unrearranged TRAV segments, between any unrearranged TRAV segment and unrearranged TRAJ segment, and between any two consecutive unrearranged TRAJ segments. In one embodiment, the non-human animal comprises a humanized TCRα locus that comprises a DNA fragment comprising a contiguous human sequence of human TRAV35 to TRAV41 and a DNA fragment comprising a contiguous human sequence of 61 human TRAJs. In one embodiment, the non-human animal comprises a humanized TCRα locus that comprises a DNA fragment comprising a contiguous human sequence of human TRAV22 to TRAV41 and a DNA fragment comprising a contiguous human sequence of 61 human TRAJs. In one embodiment, the non-human animal comprises a humanized TCRα locus that comprises a DNA fragment comprising a contiguous human sequence of human TRAV13-2 to TRAV41 and a DNA fragment comprising a contiguous human sequence of 61 human TRAJs. In one embodiment, the non-human animal comprises a humanized TCRα locus that comprises a DNA fragment comprising a contiguous human sequence of human TRAV6 to TRAV41 and 61 human TRAJs. In one embodiment, the non-human animal comprises a humanized TCRα locus that comprises a DNA fragment comprising a contiguous human sequence of human TRAV1-1 to TRAV 41 and 61 human TRAJs. In various embodiments, the DNA fragments comprising contiguous human sequences of human TCRα variable region segments also comprise restriction enzyme sites, selection cassettes, endonucleases sites, or other sites inserted to facilitate cloning and selection during the locus humanization process. In various embodiments, these additional sites do not interfere with proper functioning (e.g., rearrangement, splicing, etc.) of various genes at the TCRα locus.

In one embodiment, the humanized TCRα locus comprises 61 human Jα segments, or 100% of human Jα segments. In a particular embodiment, humanized TCRα locus comprises 8 human Vα segments and 61 human Jα segments; in another particular embodiment, humanized TCRα locus comprises 23 human Vα segments and 61 human Jα segments. In another particular embodiment, the humanized TCRα locus comprises a complete repertoire of human Vα and Jα segments, i.e., all human variable a region gene segments encoded by the α locus, or 54 human Vα and 61 human Jα segments. In various embodiments, the non-human animal does not comprise any endogenous non-human Vα or Jα segments at the TCRα locus.

The mouse TCRβ variable locus is approximately 0.6 megabases and comprises a total of 33 Vβ, 2 Dβ, and 14 Jβ segments. The human TCRβ variable locus is approximately 0.6 megabases and comprises a total of 67 Vβ, 2 Dβ, and 14 Jβ segments. In one embodiment of the invention, the genetically modified non-human animal (e.g., rodent, e.g., mouse or rat) comprises at least one human Vβ, at least one human Dβ, and at least one human Jα segment.

In one embodiment, the non-human animal comprises a humanized TCRβ locus that comprises 1, 2, 3, 4, 5, 6, 7, δ, 9, 10, 15, 20, 23, 25, 30, 35, 40, 45, 48, 50, 55, 60, or up to human 67 Vβ segments. In some embodiments, the humanized TCRβ locus comprises 8, 14, 40, 66, or human 67 Vβ segments. Thus, in some embodiments, the humanized TCRβ locus in the non-human animal may comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 99%, or 100% of human Vβ; in some embodiments, it may comprise about 20%, about 60%, about 15%, about 98%, or 100% of human Vβ.

In some embodiments, an endogenous TCRβ variable gene locus, e.g., an endogenous TCRβ mouse variable gene locus, comprises:

a replacement of one or all of the contiguous endogenous T cell variable region Vβ gene segments, e.g., one or all contiguous endogenous T cell variable region Vβ gene segments between a first 5′ trypsinogen cluster and a second 3′ trypsinogen cluster, with one or all unrearranged human T cell variable region gene segments from TRBV1 to TRBV29-1, and/or

a replacement of one or more non-contiguous endogenous Vβ gene segments (e.g., an endogenous mouse TCRBV31 gene segment) with a human TCRBV gene segment (e.g., a replacement of a mouse TCRBV31 gene segment with an orthologous human TCRBV30 gene segment).

In one embodiment, the non-human animal comprises a humanized TCRβ locus that comprises a DNA fragment comprising a contiguous human sequence of human Vβ18 to Vβ29-1 (Vβ segment is also referred to as “TRBV” or “TCRBV”). In one embodiment, the non-human animal comprises a humanized TCRβ locus that comprises a DNA fragment comprising a contiguous human sequence of human TRBV18 to TRBV29-1, a separate DNA fragment comprising a contiguous human TCRBDJ1 sequence comprising human Dβ1-Jβ1 (i.e., human Dβ1-Jβ1-1-Jβ1-6 segments), and a separate DNA fragment comprising a contiguous human TCRBDJ2 sequence that comprises human Dβ2-Jβ2 (i.e., human Dβ2-Jβ2-1-Jβ2-7 segments). An unrearranged TCRBDJ1 sequence, which may also be referred to an unrearranged TCRBJD1 cluster, comprises an unrearranged TCRBD1 segment, one to all unrearranged TCRBJ1 segments (e.g., Jβ1-1, Jβ1-2, Jβ1-3, Jβ1-4, Jβ1-5, and Jβ1-6 segments), and TCRBDJ1 non-coding sequences between the unrearranged TCRBD1 segment and the unrearranged TCRBJ1 segment and between any two consecutive unrearranged TCRBJ1 gene segments. A TCRB non-coding sequence refers to a contiguous non-coding sequence comprising non-coding recombinant signal sequences (RSSs) and other non-coding intergenic sequences found between any two consecutive unrearranged TRBV segments, and may include a TCRBDJ1 non-coding sequence, e.g., a contiguous non-coding sequence comprising non-coding recombinant signal sequences (RSSs) and other non-coding intergenic sequences found between a unrearranged TRBD1 segment and a TRBJ1 segment and between any two consecutive unrearranged TRBJ1 segments, or a TCRBDJ2 non-coding sequence, e.g., a contiguous non-coding sequence comprising non-coding recombinant signal sequences (RSSs) and other non-coding intergenic sequences found between a unrearranged TRBD2 segment and a TRBJ2 segment and between any two consecutive unrearranged TRBJ2 segments. An unrearranged TCRBDJ1 sequence may be operably linked to a plurality of unrearranged TRBV segments and a TCRBC1 constant region sequence (which may also be referred to as a TRBC1 region sequence). An unrearranged TCRBDJ2 sequence, which may also be referred to an unrearranged TCRBJD2 cluster, comprises an unrearranged TCRBD2 segment, one to all unrearranged TCRBJ2 segments (e.g., Jβ2-1, Jβ2-2, Jβ2-3, Jβ2-4, Jβ2-5, Jβ2-6, and Jβ2-7 segments), and TCRBDJ2 non-coding sequences between the unrearranged TCRBD2 segment and the unrearranged TCRBJ2 segment and between any two consecutive unrearranged TCRBJ2 gene segments. An unrearranged TCRBDJ2 sequence may be operably linked to a plurality of unrearranged TRBV segments and a TCRBC2 constant region sequence (which may also be referred to as a TRBC2 region sequence). In one embodiment, the non-human animal comprises a humanized TCRβ locus that comprises either or both (i) a TCRBDJ1 cluster wherein at least all of or at least one of Dβ1-Jβ1 segments (i.e., Dβ1, Jβ1-1, Jβ1-2, Jβ1-3, Jβ1-4, Jβ1-5, and Jβ1-6 segments) are human and wherein the non-coding sequences between the Dβ1-Jβ1 segments, including RSSs and other intergenic sequences, are non-human, e.g., mouse, optionally wherein the Dβ1 and Jβ1-1 to JβJ1-6 segments flank the same mouse TCR non-coding sequences as are normally flanked by mouse Trbd1 and mouse Trbj1-1 to Trbj1-6 segments and/or (ii) a TCRBDJ2 cluster wherein at least one of or all of the Dβ2-Jβ2 segments (i.e., Dβ2, Jβ2-1, Jβ2-2, Jβ2-3, Jβ02-3, Jβ2-4, Jβ2-5, Jβ2-6, and Jβ2-7 segments) are human and wherein the non-coding sequences between the Dβ2-Jβ2 segments, including RSSs and other intergenic sequences, are non-human, e.g., mouse, optionally wherein the Dβ2 and JγJ2-1 to Jβ2-7 gene segments flank the same mouse TCR non-coding sequences as are normally flanked by the mouse Trbd2 and mouse Trbj2-1 to Trbj2-7 gene segments. In one embodiment, the non-human animal comprises a humanized TCRβ locus that comprises a DNA fragment comprising a contiguous human sequence of human TRBV6-5 to TRBV29-1, a separate DNA fragment comprising a contiguous human sequence of human Dβ1-Jβ1 (i.e., human Dβ1-Jβ1-1-Jβ1-6 segments), and a separate DNA fragment comprising a contiguous human sequence of human Dβ2-Jβ2 (i.e., human Dβ2-Jβ2-1-Jβ2-7 segments). In one embodiment, the non-human animal comprises a humanized TCRβ locus that comprises a DNA fragment comprising a contiguous human sequence of human TRBV1 to TRBV29-1, a separate DNA fragment comprising a contiguous human sequence of human Dβ1-Jβ1, and a separate DNA fragment comprising a contiguous human sequence of human Dβ2-Jβ2. In one embodiment, the non-human animal comprises a humanized TCRβ locus that comprises a DNA fragment comprising a contiguous human sequence of human TRBV1 to TRBV29-1, a separate DNA fragment comprising a contiguous human sequence of human Dβ1-Jβ1, a separate DNA fragment comprising a contiguous human sequence of human Dβ2-Jβ2, and a separate DNA fragment comprising the sequence of human TRBV30. In various embodiments, the DNA fragments comprising contiguous human sequences of human TCRβ variable region segments also comprise restriction enzyme sites, selection cassettes, endonucleases sites, or other sites inserted to facilitate cloning and selection during the locus humanization process. In various embodiments, these additional sites do not interfere with proper functioning (e.g., rearrangement, splicing, etc.) of various genes at the TCRβ locus.

In one embodiment, the humanized TCRβ locus comprises 14 human Jβ segments, or 100% of human Jβ segments, and 2 human Dβ segments or 100% of human Dβ segments. In another embodiment, the humanized TCRβ locus comprises at least one human Vβ segment, e.g., 14 human Vβ segments, and all mouse Dβ and Jβ segments. In a particular embodiment, humanized TCRβ locus comprises 14 human Vβ segments, 2 human Dβ segments, and 14 human Jβ segments. In another particular embodiment, the humanized TCRβ locus comprises a complete repertoire of human Vβ, Dβ, and Jβ segments, i.e., all human variable β region gene segments encoded by the β locus or 67 human Vβ, 2 human Dβ, and 14 human Jβ segments. In one embodiment, the non-human animal comprises one (e.g., 5′) non-human Vβ segment at the humanized TCRβ locus. In various embodiments, the non-human animal does not comprise any endogenous non-human Vβ, Dβ, or Jβ segments at the TCRβ locus.

In one embodiment, the humanized TCRβ locus comprises 13 human Jβ segments, or 100% of functional human Jβ segments, and 2 human Dβ segments or 100% of functional human Jβ segments. In another embodiment, the humanized TCRβ locus comprises at least one human Vβ segment, e.g., 14 human Vβ segments, and all functional mouse Dβ and Jβ segments. In a particular embodiment, humanized TCRβ locus comprises 14 human Vβ segments, 2 human Dβ segments, and β functional human Jβ segments. In another particular embodiment, the humanized TCRβ locus comprises a complete repertoire of human Vβ, Dβ, and Jβ segments, i.e., all human variable β region gene segments encoded by the β locus or 67 human Vβ, 2 human Dβ, and β functional human Jβ segments. In one embodiment, the non-human animal comprises one (e.g., 5′) non-human Vβ segment at the humanized TCRβ locus. In various embodiments, the non-human animal does not comprise any endogenous non-human Vβ, Dβ, or Jβ segments at the TCRβ locus.

In various embodiments, wherein the non-human animal (e.g., rodent) comprises a repertoire of human TCRα and TCRβ (and optionally human TCRδ and TCRγ) variable region segments (e.g., a complete repertoire of variable region segments), the repertoire of various segments (e.g., the complete repertoire of various segments) is utilized by the animal to generate a diverse repertoire of TCR molecules to various antigens.

In various aspects, the non-human animals comprise contiguous portions of the human genomic TCR variable loci that comprise V, D, and J, or D and J, or V and J, or V segments arranged as in an unrearranged human genomic variable locus, e.g., comprising promoter sequences, leader sequences, intergenic sequences, regulatory sequences, etc., arranged as in a human genomic TCR variable locus. A contiguous human sequence in reference to TCRA and/or TCRB sequence(s) may also generally refer to a fully human sequence, e.g., wherein both the TCR coding sequences (e.g., TCR gene segments) and TCR non-coding sequences (e.g., non-coding DNA that separates and is flanked by TCR gene segments, such as non-coding recombination signal sequences (RSSs) and other non-coding intergenic sequences) are human, and preferably, wherein the TCR gene segments and TCR non-coding sequences are in the same sequential order in which they can be found in the human germline. As a non-limiting example, a contiguous human TCRAV sequence comprises a fully human sequence, e.g., wherein both (a) the TCRAV coding sequences (e.g., TCRAV gene segments) and (b) the TCRAV non-coding sequences (e.g., non-coding DNA that separates and is flanked by TCRAV gene segments, such as non-coding recombination signal sequences (RSSs) and other non-coding intergenic sequences) are human, and preferably, wherein the TCRAV gene segments and TCRAV non-coding sequences are in the same sequential order in which they can be found in the human germline. In another non-limiting example, a contiguous human TCRAJ sequence comprises a fully human sequence, e.g., wherein both (a) the TCRAJ coding sequences (e.g., TCRAJ gene segments) and (b) the TCRAJ non-coding sequences (e.g., non-coding DNA that separates and is flanked by TCRAJ gene segments, such as non-coding recombination signal sequences (RSSs) and other non-coding intergenic sequences) are human, and preferably, wherein the TCRJ gene segments and TCRJ non-coding sequences are in the same sequential order in which they can be found in the human germline. As another non-limiting example, a contiguous human sequence of Vβ segments may comprise a fully human sequence, e.g., wherein both the TCRBV coding sequences (e.g., TCRBV gene segments) and TCRBV non-coding sequences (e.g., non-coding DNA that separates and is flanked by TCRBV gene segments, such as non-coding recombination signal sequences (RSSs) and other non-coding intergenic sequences) are human, and preferably, wherein the TCRBV gene segments and TCRBV non-coding sequences are in the same sequential order in which they can be found in the human germline. In some embodiments, a contiguous human sequence of Dβ1-41 comprises a fully human sequence, e.g., wherein

(a) the TCRBD1 coding sequence (i.e., a TCRBDβ1 or Dβ1 segment),

(b) the TCRBJ1 (i.e., Jβ1) coding sequence(s) (i.e., a TCRBJ1-1 segment, a TCRBJ1-2 segment, a TCRBJ1-3 segment, a TCRBJ1-4 segment, a TCRBJ1-5 segment, a TCRBJ1-6 segment, and any combination thereof), and

(c) the TCRBDJ1 non-coding sequences (e.g., non-coding DNA that separates and is flanked by TCRBD1 gene segments and/or TCRBJ1 gene segments, such as non-coding recombination signal sequences (RSSs) and other non-coding intergenic sequences, e.g., a TCRBD1-TCRBJ1 non-coding sequence, a TCRBJ1-1-TCRBJ1-2 non-coding sequence, etc.)

are human, and preferably, wherein the TCRBD1 gene segments, the TCRBJ1 gene segments, and TCRBDJ1 non-coding sequences are in the same sequential order in which they can be found in the human germline. As another non-limiting example, a contiguous human sequence of Dβ2-Jβ2 comprises a fully human sequence, e.g., wherein

(a) the TCRBD2 coding sequence (i.e., a TCRBDβ2 or Dβ2 segment),

(b) the TCRBJ2 (i.e., Jβ2) coding sequence(s) (i.e., a TCRBJ2-1 segment, a TCRBJ2-2 segment, a TCRBJ2-3 segment, a TCRBJ2-4 segment, a TCRBJ2-5 segment, a TCRBJ2-6 segment, a TCRBJ2-7 segment, and any combination thereof), and

(c) the TCRBDJ2 non-coding sequences (e.g., non-coding DNA that separates and is flanked by TCRBD2 gene segments and/or TCRBJ2 gene segments, such as non-coding recombination signal sequences (RSSs) and other non-coding intergenic sequences, e.g., a TCRBD2-TCRBJ2 non-coding sequence, a TCRBJ2-1-TCRBJ2-2 non-coding sequence, etc.).

are human, and preferably, wherein the TCRBD2 gene segments, the TCRBJ2 gene segments, and TCRBDJ2 non-coding sequences are in the same sequential order in which they can be found in the human germline.

In various embodiments of the humanized TCR α,β, δ and/or γ locus, the humanized locus can comprise human coding sequences (e.g., TCR gene segments) and non-human, e.g., murine, TCR non-coding sequences, e.g., non-coding DNA that separates and is flanked by TCR gene segments such as, but not limited to, such as non-coding recombination signal sequences (RSSs) and other non-coding intergenic sequences, (e.g., a TCRAV non-coding sequence, a TCRAJ non-coding sequence, a TCRBV non-coding sequence, a TCRBD1-TCRBJ1 non-coding sequence, a TCRBJ1 non-coding sequence (e.g., a TCRBJ1-1-TCRBJ1-2 non-coding sequence, e.g., a TCRBJ1-2-TCRBJ1-3 non-coding sequence, etc.), a TCRBD2-TCRBJ2 non-coding sequence, a TCRBJ2 non-coding sequence (e.g., TCRBJ2-1-TCRBJ2-2 noncoding sequence, a TCRBJ2-2-TCRBJ2-3 non-coding sequence, etc.)). In some embodiments, the human TCR gene segments replace orthologous non-human (e.g., mouse) TCR gene segments such that the human TCR gene segments flank the same non-human (e.g., mouse) TCR non-coding sequences as those that are flanked by the replaced orthologous non-human (e.g., mouse) TCR gene segments, e.g., such that the human TCR gene segments and non-human (e.g., mouse) TCR non-coding sequences are in the same order as that would be found in the non-human (e.g., mouse) germline but for the replacements of the orthologous (e.g., non-human) TCR gene segments. See, e.g., FIG. 4C.

In other aspects, the various segments are arranged as in an unrearranged non-human genomic TCR variable locus. In various embodiments of the humanized TCR α, β, δ and/or γ locus, the humanized locus can comprise two or more human genomic segments that do not appear in a human genome juxtaposed, e.g., a fragment of V segments of the human variable locus located in a human genome proximal to the constant region, juxtaposed with a fragment of V segments of the human variable locus located in a human genome at the upstream end of the human variable locus.

In both mouse and human, the TCRδ gene segments are located with the TCRα locus (see FIG. 4A, top, TCRD region boxed). TCRδ J and D segments are located between Vα and Jα segments, while TCRδ V segments are interspersed throughout the TCRα locus, with the majority located among various Vα segments. The number and locations of various TCRδ segments can be determined from the IMGT database. Due to the genomic arrangement of TCRδ gene segments within the TCRα locus, successful rearrangement at the TCRα locus may delete or inactivate the TCRδ gene segments.

In some embodiments of the invention, a non-human animal comprising an unrearranged human TCRα variable gene locus also comprises at least one human Vδ segment, e.g., up to complete repertoire of human Vδ segments. Thus, in some embodiments, the replacement of endogenous TCRα variable gene locus results in a replacement of at least one non-human Vδ segment with a human Vδ segment. In other embodiments, the non-human animal of the invention comprises a complete repertoire of human Vδ, Dδ, and Jδ segments at the unrearranged humanized TCRα locus; in yet other embodiments, the non-human animal comprises a complete unrearranged human TCRδ locus at the unrearranged humanized TCRα locus (i.e., a TCRδ locus including human variable region segments, as well as human enhancer and constant region). An exemplary embodiment for constructing an unrearranged humanized TCRα locus comprising complete unrearranged TCRδ locus is depicted in U.S. Pat. No. 9,113,616, incorporated herein by reference.

In yet another embodiment, the non-human animal of the invention further comprises an unrearranged humanized TCRγ locus, e.g., a TCRγ locus comprising at least one human Vγ and at least one human Jγ segments (e.g., a complete repertoire of human Vγ and human Jγ variable region segments). The human TCRγ locus is on human chromosome 7, while the mouse TCRγ locus is on mouse chromosome 13. See the IMGT database for more detail on the TCRγ locus.

In one aspect, the non-human animal (e.g., rodent, e.g., mouse or rat) comprising humanized TCRα and β variable gene loci (and, optionally humanized TCRδ/γ variable gene loci) described herein expresses a humanized T cell receptor comprising a human variable region and a non-human (e.g., rodent, e.g., mouse or rat) constant region on a surface of a T cell. In some aspects, the non-human animal is capable or expressing a diverse repertoire of humanized T cell receptors that recognize a variety of presented antigens.

In one aspect, the non-human animal (e.g., rodent, e.g., mouse or rat) comprising a humanized TCRβ variable gene locus that comprises non-human animal (e.g., rodent, e.g., mouse or rat) TCRB non-coding sequences as described herein comprises a population of spleen cells, e.g., CD4+ and/or CD8+ T cells, of which at least 10% of the TCR expressed by the population of spleen cells is derived from gene segments from the TCRBDJ1 cluster and at least 10% of the TCR expressed by the population of spleen cells is derived from gene segments from the TCRBDJ2 cluster. In some embodiments, the non-human animal (e.g., rodent, e.g., mouse or rat) comprising a humanized TCRβ variable gene locus that comprises non-human animal (e.g., rodent, e.g., mouse or rat) TCRB non-coding sequences as described herein comprises a population of spleen cells, e.g., CD4+ and/or CD8+ T cells, of which at least 15% of the TCR expressed by the population of spleen cells is derived from gene segments from the TCRBDJ1 cluster and at least 15% of the TCR expressed by the population of spleen cells is derived from gene segments from the TCRBDJ2 cluster. In some embodiments, the non-human animal (e.g., rodent, e.g., mouse or rat) comprising a humanized TCRβ variable gene locus that comprises non-human animal (e.g., rodent, e.g., mouse or rat) TCRB non-coding sequences as described herein comprises a population of spleen cells, e.g., CD4+ and/or CD8+ T cells, of which at least 20% of the TCR expressed by the population of spleen cells is derived from gene segments from the TCRBDJ1 cluster and at least 20% of the TCR expressed by the population of spleen cells is derived from gene segments from the TCRBDJ2 cluster. In some embodiments, the non-human animal (e.g., rodent, e.g., mouse or rat) comprising a humanized TCRβ variable gene locus that comprises non-human animal (e.g., rodent, e.g., mouse or rat) TCRB non-coding sequences as described herein comprises a population of spleen cells, e.g., CD4+ and/or CD8+ T cells, of which at least 30% of the TCR expressed by the population of spleen cells is derived from gene segments from the TCRBDJ1 cluster and at least 30% of the TCR expressed by the population of spleen cells is derived from gene segments from the TCRBDJ2 cluster. In some embodiments, the non-human animal (e.g., rodent, e.g., mouse or rat) comprising a humanized TCRβ variable gene locus that comprises non-human animal (e.g., rodent, e.g., mouse or rat) TCRB non-coding sequences as described herein comprises a population of spleen cells, e.g., CD4+ and/or CD8+ T cells, of which at least 40% of the TCR expressed by the population of spleen cells is derived from gene segments from the TCRBDJ2 cluster. In some embodiments, the non-human animal (e.g., rodent, e.g., mouse or rat) comprising a humanized TCRβ variable gene locus that comprises non-human animal (e.g., rodent, e.g., mouse or rat) TCRB non-coding sequences as described herein comprises a population of spleen cells, e.g., CD4+ and/or CD8+ T cells, of which at least 50% of the TCR expressed by the population of spleen cells is derived from gene segments from the TCRBDJ2 cluster. In some embodiments, the non-human animal (e.g., rodent, e.g., mouse or rat) comprising a humanized TCRβ variable gene locus that comprises non-human animal (e.g., rodent, e.g., mouse or rat) TCRB non-coding sequences as described herein comprises a population of spleen cells, e.g., CD4+ and/or CD8+ T cells, of which at least 60% of the TCR expressed by the population of spleen cells is derived from gene segments from the TCRBDJ2 cluster. In some embodiments, the non-human animal (e.g., rodent, e.g., mouse or rat) comprising a humanized TCRβ variable gene locus that comprises non-human animal (e.g., rodent, e.g., mouse or rat) TCRB non-coding sequences as described herein comprises a population of spleen cells, e.g., CD4+ and/or CD8+ T cells, of which at least 70% of the TCR expressed by the population of spleen cells is derived from gene segments from the TCRBDJ2 cluster. In some embodiments, the non-human animal (e.g., rodent, e.g., mouse or rat) comprising a humanized TCRβ variable gene locus that comprises non-human animal (e.g., rodent, e.g., mouse or rat) TCRB non-coding sequences as described herein comprises a population of spleen cells, e.g., a population of CD4+ and/or CD8+ T cells, which expresses TCRs derived from gene segments from the TCRBDJ1 cluster and TCRs derived from gene segments from the TCRBDJ2 cluster at a ratio of 1:3, 3:7, 1:2, 2:3, or 1:1. In some embodiments, the non-human animal (e.g., rodent, e.g., mouse or rat) comprising a humanized TCRβ variable gene locus that comprises non-human animal (e.g., rodent, e.g., mouse or rat) TCRB non-coding sequences as described herein comprises a population of spleen cells, e.g., a population of CD4+ and/or CD8+ T cells, which expresses TCRs derived from gene segments from the TCRBDJ2 cluster and TCRs derived from gene segments from the TCRBDJ1 cluster at a ratio of 1:3, 3:7, 1:2, or 2:3.

In various embodiments of the invention, the humanized T cell receptor polypeptides described herein comprise human leader sequences. In alternative embodiments, the humanized TCR receptor nucleic acid sequences are engineered such that the humanized TCR polypeptides comprise non-human leader sequences.

The humanized TCR polypeptides described herein may be expressed under control of endogenous non-human regulatory elements (e.g., rodent regulatory elements), e.g., promoter, silencer, enhancer, etc. The humanized TCR polypeptides described herein may alternatively be expressed under control of human regulatory elements. In various embodiments, the non-human animals described herein further comprise all regulatory and other sequences normally found in situ in the human genome.

In various embodiments, the human variable region of the humanized TCR protein is capable of interacting with various proteins on the surface of the same cell or another cell. In one embodiment, the human variable region of the humanized TCR interacts with MHC proteins (e.g., MHC class I or II proteins) presenting antigens on the surface of the second cell, e.g., an antigen presenting cell (APC). In some embodiments, the MHC I or II protein is a non-human (e.g., rodent, e.g., mouse or rat) protein. In other embodiments, the MHC I or II protein is a human(ized) protein. In one aspect, the second cell, e.g., the APC, is an endogenous non-human cell expressing a human or humanized MHC molecule. In a different embodiment, the second cell is a human cell expressing a human MHC molecule.

In one aspect, the non-human animal expresses a humanized T cell receptor with a non-human constant region on the surface of a T cell, wherein the receptor is capable of interacting with non-human molecules, e.g., anchor or signaling molecules expressed in the T cell (e.g., CD3 molecules, the ζ chain, or other proteins anchored to the TCR through the CD3 molecules or the ζ chain). Thus, in one aspect, a cellular complex is provided, comprising (a) a non-human T-cell that expresses (i) a TCR that comprises a humanized TCRα chain as described herein and humanized TCRβ chain as described herein and (ii) a chimeric co-receptor as described herein and (b) a non-human antigen-presenting cell comprising an antigen bound to a chimeric MHC I and/or chimeric MHC II as described herein. In one embodiment, the non-human constant TCRα and TCRβ chains are complexed with a non-human zeta (ζ) chain homodimer and CD3 heterodimers. In one embodiment, the cellular complex is an in vivo cellular complex. In one embodiment, the cellular complex is an in vitro cellular complex.

In various embodiments, the non-human animals (e.g., rodents, e.g., mice or rats) described herein produce T cells that are capable of undergoing thymic development, progressing from DN1 to DN2 to DN3 to DN4 to DP and to CD4 or CD8 SP T cells. Such T cells of the non-human animal of the invention express cell surface molecules typically produced by a T cell during a particular stage of thymic development (e.g., CD25, CD44, Kit, CD3, pTα, etc.). Thus, in one embodiment, the non-human animals described herein may express pTα complexed with TCRβ at the DN3 stage of thymic development. The non-human animals described herein express T cells capable of undergoing thymic development to produce CD4+ and CD8+ T cells.

In various embodiments, the non-human animals described herein produce T cells that are capable of undergoing T cell differentiation in the periphery. In some embodiments, the non-human animals described herein are capable of producing a repertoire of effector T cells, e.g., CTL (cytotoxic T lymphocytes), T_(H)1, T_(H)2, T_(REG), T_(H)17, etc. Thus, in these embodiments, the non-human animals described herein generate effector T cells that fulfill different functions typical of the particular T cell type, e.g., recognize, bind, and respond to foreign antigens. In various embodiments, the non-human animals described herein produce effector T cells that kill cells displaying peptide fragments of cytosolic pathogens expressed in the context of MHC I molecules; recognize peptides derived from antigens degraded in intracellular vesicles and presented by MHC II molecules on the surface of macrophages and induce macrophages to kill microorganisms; produce cytokines that drive B cell differentiation; activate B cells to produce opsonizing antibodies; induce epithelial cells to produce chemokines that recruit neutrophils to infection sites; etc.

In additional embodiments, the non-human animals described herein comprise CD3+ T cells in the periphery, e.g., in the spleen. In other aspects, the non-human animals described herein are capable of generating a population of memory T cells in response an antigen of interest. For example, the non-human animals generate both central memory T cells (Tcm) and effector memory T cells (Tem) to an antigen, e.g., antigen of interest (e.g., antigen being tested for vaccine development, etc.).

DN1 and DN2 cells that do not receive sufficient signals (e.g., Notch signals) may develop into B cells, myeloid cells (e.g., dendritic cells), mast cells and NK cells. See, e.g., Yashiro-Ohtani et al. (2010) Notch regulation of early thymocyte development, Seminars in Immunology 22:261-69. In some embodiments, the non-human animals described herein develop B cells, myeloid cells (e.g., dendritic cells), mast cells and NK cells. In some embodiments, the non-human animals described herein develop a dendritic cell population in the thymus.

The predominant type of T cell receptors expressed on the surface of T cells is TCRα/β, with the minority of the cells expressing TCRδ/γ. In some embodiments of the invention, the T cells of the non-human animals comprising humanized TCRα and/or β loci exhibit utilization of TCRα/β and TCRδ/γ loci, e.g., utilization of TCRα/β and TCRδ/γ loci that is similar to the wild type animal (e.g., the T cells of the non-human animals described herein express TCRα/β and TCRδ/γ proteins in comparable proportions to that expressed by wild type animals). Thus, in some embodiments, the non-human animals comprising humanized TCRα/β and endogenous non-human TCRδ/γ loci exhibit utilization of all loci.

Human or Humanized MHC Molecules

In various embodiments, provided herein are genetically modified non-human animals that co-express at least one humanized T cell co-receptor, at least one humanized MHC that associates with the humanized T cell co-receptor, and optionally, a humanized TCR, which upon recognizing and binding peptide presented by the humanized MHC, and in conjunction with the humanized co-receptor, provides activation signals to the cell expressing the humanized TCR and chimeric T cell co-receptor polypeptides. Accordingly, a non-human animal as disclosed herein comprises at least one of a first, second, and/or third nucleic acid sequence, each of which encodes a different human or humanized MHC polypeptide selected from the group consisting of a human or humanized MHC II α polypeptide, a human or humanized MHC II β polypeptide, and a human or humanized MHC I α polypeptide; the non-human animal also optionally comprises a human or humanized β2 microglobulin. Use of the first, second, and third designations herein is not to be construed as limiting the non-human animals disclosed herein as requiring all three nucleic acid sequences or the presence of any of the human or humanized MHC polypeptides in any specific order.

Accordingly, in some embodiments, a non-human animal as disclosed herein may comprise, e.g., a first and second nucleotide sequence encoding e.g., a human or chimeric CD8α polypeptide and a human or chimeric CD8β polypeptide, an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCRα constant gene sequence and/or an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCRβ constant gene sequence, and optionally a first and second nucleic acid sequence encoding, e.g., a human or humanized MHC I α polypeptide and a human or humanized β2-microglobulin polypeptide. In other embodiments, a non-human animal as disclosed herein may comprise, e.g., a first nucleotide sequence encoding, e.g., a chimeric CD4 polypeptide; an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCRα constant gene sequence and/or an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCRβ constant gene sequence; and optionally a first and second nucleic acid sequence encoding, e.g., a human or humanized MHC II α polypeptide and a human or humanized MHC II β polypeptide. In some embodiment, a non-human animal as disclosed herein may comprise, e.g., a first, second and third nucleotide sequence encoding e.g., a chimeric CD4 polypeptide, a chimeric CD8α polypeptide, and a chimeric CD8β polypeptide; an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCRα constant gene sequence and/or an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCRβ constant gene sequence; and optionally a first, second, third and fourth nucleic acid sequence encoding, e.g., a human or humanized MHC II α polypeptide, a human or humanized MHC II β polypeptide, a human or humanized MHC I α polypeptide, and a human or humanized a β2-microglobulin polypeptide.

In various embodiments, provided herein is a genetically modified non-human animal, e.g., rodent (e.g., mouse or rat) comprising in its genome a nucleic acid sequence encoding a human or humanized MHC I polypeptide and/or a nucleic acid sequence encoding human or humanized MHC II protein. The MHC I nucleic acid sequence may encode an MHC I polypeptide that is partially human and partially non-human, e.g., chimeric human/non-human MHC I polypeptide, and the MHC II nucleic acid sequence may encode an MHC II protein that is partially human and partially non-human, e.g., chimeric human/non-human MHC II protein (e.g., comprising chimeric human/non-human MHC II α and β polypeptides). In some aspects, the animal does not express endogenous MHC I and/or endogenous MHC II polypeptides, e.g., functional endogenous MHC I and/or MHC II polypeptides on a cell surface. In some embodiments, the only MHC I and/or MHC II molecules expressed on a cell surface of the animal are chimeric MHC I and/or MHC II molecules.

A genetically modified non-human animal comprising in its genome, e.g., at the endogenous locus, a nucleic acid sequence encoding a chimeric human/non-human MHC I polypeptide is disclosed in U.S. Patent Publication Nos. 20130111617 and 20130185819, which publications are incorporated herein by reference in their entireties. A genetically modified non-human animal comprising in its genome, e.g., at the endogenous locus, a nucleic acid sequence encoding humanized, e.g., chimeric human/non-human MHC II polypeptides is disclosed in U.S. Pat. No. 8,847,005 and in U.S. Patent Publication No 20130185820, each of which are incorporated herein by reference in their entireties. A genetically modified non-human animal comprising in its genome, e.g., at the endogenous locus, a nucleic acid sequence encoding a chimeric human/non-human MHC I polypeptide and comprising in its genome, e.g., at the endogenous locus, a nucleic acid sequence encoding humanized, e.g., chimeric human/non-human MHC II polypeptides, is disclosed in U.S. Patent Publication No. 20140245467, which is incorporated herein by reference in its entirety.

In various embodiments provided herein is a genetically modified non-human animal comprising in its genome, e.g., at one or more endogenous MHC loci, a first nucleic acid sequence encoding a chimeric human/non-human MHC I polypeptide, wherein a human portion of the chimeric MHC I polypeptide comprises an extracellular portion (or part thereof, e.g., one or more extracellular domains) of a human MHC I polypeptide; a second nucleic acid sequence encoding a chimeric human/non-human MHC II α polypeptide, wherein a human portion of the chimeric MHC II α polypeptide comprises an extracellular portion (or part thereof, e.g., one or more extracellular domains) of a human MHC II α polypeptide; and/or a third nucleic acid sequence encoding a chimeric human/non-human MHC II β polypeptide, wherein a human portion of the chimeric MHC II β polypeptide comprises an extracellular portion (or part thereof, e.g., one or more extracellular domains) of a human MHC II β polypeptide; wherein the non-human animal expresses functional chimeric human/non-human MHC I and MHC II proteins from its endogenous non-human MHC locus. In one embodiment, the first, second, and/or third nucleic acid sequences are respectively located the endogenous non-human MHC I, MHC II α and MHC II β loci. In one embodiment, wherein the non-human animal is a mouse, the first, second, and/or third nucleic acid sequences are located at the endogenous mouse MHC locus on mouse chromosome 17. In one embodiment, the first nucleic acid sequence is located at the endogenous non-human MHC I locus. In one embodiment, the second nucleic acid sequence is located at the endogenous non-human MHC II α locus. In one embodiment, the third nucleic acid sequence is located at the endogenous non-human MHC II β locus.

In one embodiment, the non-human animal only expresses the chimeric human/non-human MHC I, MHC II α and/or MHC β II polypeptides and does not express endogenous non-human MHC polypeptides (e.g., functional endogenous MHC I, II a and/or II β polypeptides) from the endogenous non-human MHC locus. In one embodiment, the animal described herein expresses a functional chimeric MHC I and a functional chimeric MHC II on the surface of its cells, e.g., antigen presenting cells, etc. In one embodiment, the only MHC I and MHC II expressed by the animal on a cell surface are chimeric MHC I and chimeric MHC II, and the animal does not express any endogenous MHC I and MHC II on a cell surface.

In one embodiment, the chimeric human/non-human MHC I polypeptide comprises in its human portion a peptide binding cleft, e.g., of a human MHC I polypeptide. In one aspect, the human portion of the chimeric polypeptide comprises an extracellular portion of a human MHC I. In this embodiment, the human portion of the chimeric polypeptide comprises an extracellular domain of an α chain of a human MHC I. In one embodiment, the human portion of the chimeric polypeptide comprises α1 and α2 domains of a human MHC I. In another embodiment, the human portion of the chimeric polypeptide comprises α1, α2, and α3 domains of a human MHC I.

In one aspect, a human portion of the chimeric MHC II α polypeptide and/or a human portion of the chimeric MHC II β polypeptide comprises a peptide-binding domain of a human MHC II α polypeptide and/or human MHC II β polypeptide, respectively. In one aspect, a human portion of the chimeric MHC II α and/or β polypeptide comprises an extracellular portion of a human MHC II α and/or β polypeptide, respectively. In one embodiment, a human portion of the chimeric MHC II α polypeptide comprises α1 domain of a human MHC II α polypeptide; in another embodiment, a human portion of the chimeric MHC II α polypeptide comprises α1 and α2 domains of a human MHC II α polypeptide. In an additional embodiment, a human portion of the chimeric MHC II β polypeptide comprises β1 domain of a human MHC II β polypeptide; in another embodiment, a human portion of the chimeric MHC II β polypeptide comprises β1 and β2 domains of a human MHC II β polypeptide.

In some embodiments, the human or humanized MHC I polypeptide may be derived from a functional human HLA molecule encoded by any of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G loci. The human or humanized MHC II polypeptide may be derived from a functional human HLA molecule encoded by an of HLA-DP, -DQ, and -DR loci. A list of commonly used HLA antigens and alleles is described in Shankarkumar et al. ((2004) The Human Leukocyte Antigen (HLA) System, Int. J. Hum. Genet. 4(2):91-103), incorporated herein by reference. Shankarkumar et al. also present a brief explanation of HLA nomenclature used in the art. Additional information regarding HLA nomenclature and various HLA alleles can be found in Holdsworth et al. (2009) The HLA dictionary 2008: a summary of HLA-A, -B, -C, -DRB1/3/4/5, and DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR, and -DQ antigens, Tissue Antigens 73:95-170, and a recent update by Marsh et al. (2010) Nomenclature for factors of the HLA system, 2010, Tissue Antigens 75:291-455, both incorporated herein by reference. In some embodiments, the MHC I or MHC II polypeptides may be derived from any functional human HLA-A, B, C, DR, or DQ molecules. Thus, the human or humanized MHC I and/or II polypeptides may be derived from any functional human HLA molecules described therein. In some embodiments, all MHC I and MHC II polypeptides expressed on a cell surface comprise a portion derived from human HLA molecules.

Of particular interest are human HLA molecules, specific polymorphic HLA alleles, known to be associated with a number of human diseases, e.g., human autoimmune diseases. In fact, specific polymorphisms in HLA loci have been identified that correlate with development of rheumatoid arthritis, type I diabetes, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, Graves' disease, systemic lupus erythematosus, celiac disease, Crohn's disease, ulcerative colitis, and other autoimmune disorders. See, e.g., Wong and Wen (2004) What can the HLA transgenic mouse tell us about autoimmune diabetes?, Diabetologia 47:1476-87; Taneja and David (1998) HLA Transgenic Mice as Humanized Mouse Models of Disease and Immunity, J. Clin. Invest. 101:921-26; Bakker et al. (2006), A high-resolution HLA and SNP haplotype map for disease association studies in the extended human MHC, Nature Genetics 38:1166-72 and Supplementary Information; and International MHC and Autoimmunity Genetics Network (2009) Mapping of multiple susceptibility variants within the MHC region for 7 immune-mediated diseases, Proc. Natl. Acad. Sci. USA 106:18680-85. Thus, the human or humanized MHC I and/or II polypeptides may be derived from a human HLA molecule known to be associated with a particular disease, e.g., autoimmune disease.

In one specific aspect, the human or humanized MHC I polypeptide is derived from human HLA-A. In a specific embodiment, the HLA-A polypeptide is an HLA-A2 polypeptide (e.g., and HLA-A2.1 polypeptide). In one embodiment, the HLA-A polypeptide is a polypeptide encoded by an HLA-A*0201 allele, e.g., HLA-A*02:01:01:01 allele. The HLA-A*0201 allele is commonly used amongst the North American population. Although the present Examples describe this particular HLA sequence, any suitable HLA-A sequence is encompassed herein, e.g., polymorphic variants of HLA-A2 exhibited in human population, sequences with one or more conservative or non-conservative amino acid modifications, nucleic acid sequences differing from the sequence described herein due to the degeneracy of genetic code, etc.

In another specific aspect, the human portion of the chimeric MHC I polypeptide is derived from human MHC I selected from HLA-B and HLA-C. In one aspect, it is derived from HLA-B, e.g., HLA-B27. In another aspect, it is derived from HLA-A3, -B7, -Cw6, etc.

In one specific aspect, the human portions of the humanized MHC II α and β polypeptides described herein are derived from human HLA-DR, e.g., HLA-DR2. Typically, HLA-DR α chains are monomorphic, e.g., the α chain of HLA-DR complex is encoded by HLA-DRA gene (e.g., HLA-DRα*01 gene). On the other hand, the HLA-DR β chain is polymorphic. Thus, HLA-DR2 comprises an α chain encoded by HLA-DRA gene and a β chain encoded by HLA-DR1β*1501 gene. Although the present Examples describe these particular HLA sequences; any suitable HLA-DR sequences are encompassed herein, e.g., polymorphic variants exhibited in human population, sequences with one or more conservative or non-conservative amino acid modifications, nucleic acid sequences differing from the sequences described herein due to the degeneracy of genetic code, etc.

The human portions of the chimeric MHC II α and/or β polypeptide may be encoded by nucleic acid sequences of HLA alleles known to be associated with common human diseases. Such HLA alleles include, but are not limited to, HLA-DRB1*0401, -DRB1*0301, -DQA1*0501, -DQB1*0201, DRB1*1501, -DRB1*1502, -DQB1*0602, -DQA1*0102, -DQA1*0201, -DQB1*0202, -DQA1*0501, and combinations thereof. For a summary of HLA allele/disease associations, see Bakker et al. (2006), supra, incorporated herein by reference.

In one aspect, the non-human portion of a chimeric human/non-human MHC I, MHC II α and/or MHC II β polypeptide(s) comprises transmembrane and/or cytoplasmic domains of an endogenous non-human (e.g., rodent, e.g., mouse, rat, etc.) MHC I, MHC II α and/or MHC II β polypeptide(s), respectively. Thus, the non-human portion of the chimeric human/non-human MHC I polypeptide may comprise transmembrane and/or cytoplasmic domains of an endogenous non-human MHC I polypeptide. The non-human portion of a chimeric MHC II α polypeptide may comprise transmembrane and/or cytoplasmic domains of an endogenous non-human MHC II α polypeptide. The non-human portion of a chimeric human/non-human MHC II β polypeptide may comprise transmembrane and/or cytoplasmic domains of an endogenous non-human MHC II β polypeptide. In one aspect, the non-human animal is mouse, and a non-human portion of the chimeric MHC I polypeptide is derived from a mouse H-2K protein. In one aspect, the animal is a mouse, and non-human portions of the chimeric MHC II α and β polypeptides are derived from a mouse H-2E protein. Thus, a non-human portion of the chimeric MHC I polypeptide may comprise transmembrane and cytoplasmic domains derived from a mouse H-2K, and non-human portions of the chimeric MHC II α and β polypeptides may comprise transmembrane and cytoplasmic domains derived from a mouse H-2E protein. Although specific H-2K and H-2E sequences are contemplated in the Examples, any suitable sequences, e.g., polymorphic variants, conservative/non-conservative amino acid substitutions, etc., are encompassed herein. In one aspect, the non-human animal is a mouse, and the mouse does not express functional endogenous MHC polypeptides from its H-2D locus. In some embodiments, the mouse is engineered to lack all or a portion of an endogenous H-2D locus. In other aspects, the mouse does not express any functional endogenous mouse MHC I and MHC II on a cell surface.

A chimeric human/non-human polypeptide may be such that it comprises a human or a non-human leader (signal) sequence. In one embodiment, the chimeric MHC I polypeptide comprises a non-human leader sequence of an endogenous MHC I polypeptide. In one embodiment, the chimeric MHC II α polypeptide comprises a non-human leader sequence of an endogenous MHC II α polypeptide. In one embodiment, the chimeric MHC II β polypeptide comprises a non-human leader sequence of an endogenous MHC II β polypeptide. In an alternative embodiment, the chimeric MHC I, MHC II α and/or MHC II β polypeptide(s) comprises a non-human leader sequence of MHC I, MHC II α and/or MHC II polypeptide(s), respectively, from another non-human animal, e.g., another rodent or another mouse strain. Thus, the nucleic acid sequence encoding the chimeric MHC I, MHC II α and/or MHC II β polypeptide may be operably linked to a nucleic acid sequence encoding a non-human MHC I, MHC II α and/or MHC II β leader sequence, respectively. In yet another embodiment, the chimeric MHC I, MHC II α and/or MHC II β polypeptide(s) comprises a human leader sequence of human MHC I, human MHC II α and/or human MHC II β polypeptide, respectively (e.g., a leader sequence of human HLA-A2, human HLA-DRα and/or human HLA-DRβ1*1501, respectively).

A chimeric human/non-human MHC I, MHC II α and/or MHC II β polypeptide may comprise in its human portion a complete or substantially complete extracellular domain of a human MHC I, human MHC II α and/or human MHC II β polypeptide, respectively. Thus, a human portion may comprise at least 80%, preferably at least 85%, more preferably at least 90%, e.g., 95% or more of the amino acids encoding an extracellular domain of a human MHC I, human MHC II α and/or human MHC II β polypeptide (e.g., human HLA-A2, human HLA-DRα and/or human HLA-DRβ1*1501). In one example, substantially complete extracellular domain of the human MHC I, human MHC II α and/or human MHC II polypeptide lacks a human leader sequence. In another example, the chimeric human/non-human MHC I, chimeric human/non-human MHC II α and/or the chimeric human/non-human MHC II β polypeptide comprises a human leader sequence.

Moreover, the chimeric MHC I, MHC II α and/or MHC II β polypeptide may be operably linked to (e.g., be expressed under the regulatory control of) endogenous non-human promoter and regulatory elements, e.g., mouse MHC I, MHC II α and/or MHC II β regulatory elements, respectively. Such arrangement will facilitate proper expression of the chimeric MHC I and/or MHC II polypeptides in the non-human animal, e.g., during immune response in the non-human animal.

In a further embodiment, a non-human animal of the invention, e.g., a rodent, e.g., a mouse, comprises (e.g., at an endogenous β2 microglobulin locus) a nucleic acid sequence encoding a human or humanized β2 microglobulin. β2 microglobulin or the light chain of the MHC class I complex (also abbreviated “(β2M”) is a small (12 kDa) non-glycosylated protein, that functions primarily to stabilize the MHC I α chain. Generation of human or humanized β2 microglobulin animals is described in detail in U.S. Patent Publication No. 20130111617, and is incorporated herein by reference.

The nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide may comprise nucleic acid residues corresponding to the entire human β2 microglobulin gene. Alternatively, the nucleotide sequence may comprise nucleic acid residues encoding amino acid sequence set forth in amino acids 21-119 of a human β2 microglobulin protein (i.e., amino acid residues corresponding to the mature human β2 microglobulin). In an alternative embodiment, the nucleotide sequence may comprise nucleic acid residues encoding amino acid sequence set forth in amino acids 23-115 of a human β2 microglobulin protein, for example, amino acid sequence set forth in amino acids 23-119 of a human β2 microglobulin protein. The nucleic and amino acid sequences of human β2 microglobulin are described in Gussow et al., supra, incorporated herein by reference.

Thus, the human or humanized β2 microglobulin polypeptide may comprise amino acid sequence set forth in amino acids 23-115 of a human β2 microglobulin polypeptide, e.g., amino acid sequence set forth in amino acids 23-119 of a human β2 microglobulin polypeptide, e.g., amino acid sequence set forth in amino acids 21-119 of a human β2 microglobulin polypeptide. Alternatively, the human β2 microglobulin may comprise amino acids 1-119 of a human β2 microglobulin polypeptide.

In some embodiments, the nucleotide sequence encoding a human or humanized β2 microglobulin comprises a nucleotide sequence set forth in exon 2 to exon 4 of a human β2 microglobulin gene. Alternatively, the nucleotide sequence comprises nucleotide sequences set forth in exons 2, 3, and 4 of a human β2 microglobulin gene. In this embodiment, the nucleotide sequences set forth in exons 2, 3, and 4 are operably linked to allow for normal transcription and translation of the gene. Thus, in one embodiment, the human sequence comprises a nucleotide sequence corresponding to exon 2 to exon 4 of a human β2 microglobulin gene. In a specific embodiment, the human sequence comprises a nucleotide sequence corresponding to exon 2 to about 267 bp after exon 4 of a human β2 microglobulin gene. In a specific embodiment, the human sequence comprises about 2.8 kb of a human β2 microglobulin gene.

Thus, the human or humanized β2 microglobulin polypeptide may be encoded by a nucleotide sequence comprising nucleotide sequence set forth in exon 2 to exon 4 of a human β2 microglobulin, e.g., nucleotide sequence corresponding to exon 2 to exon 4 of a human β2 microglobulin gene. Alternatively, the polypeptide may be encoded by a nucleotide sequence comprising nucleotide sequences set forth in exons 2, 3, and 4 of a human β2 microglobulin gene. In a specific embodiment, the human or humanized β2 microglobulin polypeptide is encoded by a nucleotide sequence corresponding to exon 2 to about 267 bp after exon 4 of a human β2 microglobulin gene. In another specific embodiment, the human or humanized polypeptide is encoded by a nucleotide sequence comprising about 2.8 kb of a human β2 microglobulin gene. As exon 4 of the β2 microglobulin gene contains the 5′ untranslated region, the human or humanized polypeptide may be encoded by a nucleotide sequence comprising exons 2 and 3 of the β2 microglobulin gene.

It would be understood by those of ordinary skill in the art that although specific nucleic acid and amino acid sequences to generate genetically engineered animals are described herein, sequences of one or more conservative or non-conservative amino acid substitutions, or sequences differing from those described herein due to the degeneracy of the genetic code, are also provided.

Therefore, a non-human animal that expresses a human β2 microglobulin sequence is provided, wherein the β2 microglobulin sequence is at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human β2 microglobulin sequence. In a specific embodiment, the β2 microglobulin sequence is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to the human β2 microglobulin sequence described herein. In one embodiment, the human β2 microglobulin sequence comprises one or more conservative substitutions. In one embodiment, the human β2 microglobulin sequence comprises one or more non-conservative substitutions.

In addition, provided are non-human animals wherein the nucleotide sequence encoding a human or humanized β2 microglobulin protein also comprises a nucleotide sequence set forth in exon 1 of a non-human β2 microglobulin gene. Thus, in a specific embodiment, the non-human animal comprises in its genome a nucleotide sequence encoding a human or humanized β2 microglobulin wherein the nucleotide sequence comprises exon 1 of a non-human β2 microglobulin and exons 2, 3, and 4 of a human β2 microglobulin gene. Thus, the human or humanized β2 microglobulin polypeptide is encoded by exon 1 of a non-human β2 microglobulin gene and exons 2, 3, and 4 of a human β2 microglobulin gene (e.g., exons 2 and 3 of a human β2 microglobulin gene).

In one embodiment, the non-human animal (e.g., rodent, e.g., mouse) of the invention, in addition to a nucleotide sequence encoding a chimeric CD8 protein, further comprises a nucleic acid sequence encoding a human or humanized MHC I protein, such that the chimeric CD8 protein expressed on the surface of a T cell of the animal is capable of associating, binding and/or interacting with a human or humanized MHC I expressed on a surface of a second cell, e.g., an antigen presenting cell. In one embodiment, the MHC I protein comprises an extracellular domain of a human MHC I polypeptide. In one embodiment, the animal further comprises a human or humanized β2 microglobulin polypeptide. Exemplary genetically modified animals expressing a human or humanized MHC I polypeptide and/or β2 microglobulin polypeptide are described in U.S. Patent Publication Nos. 20130111617 and 20130185819, both incorporated herein by reference in their entireties. Thus, in one embodiment, the animal comprising chimeric CD8 protein described herein may further comprise a humanized MHC I complex, wherein the humanized MHC I complex comprises: (1) a humanized MHC I polypeptide, e.g., wherein the humanized MHC I polypeptide comprises a human MHC I extracellular domain and transmembrane and cytoplasmic domains of an endogenous (e.g., mouse) MHC I, e.g., wherein the humanized MHC I comprises α1, α2, and α3 domains of a human MHC I polypeptide, and (2) a human or humanized β2 microglobulin polypeptide (e.g., the animal comprises in its genome a nucleotide sequence set forth in exons 2, 3, and 4 of a human β2 microglobulin). In one aspect, both humanized MHC I and human or humanized β2 microglobulin polypeptides are encoded by nucleotide sequences located at endogenous MHC I and β2 microglobulin loci, respectively; in one aspect, the animal does not express functional endogenous MHC I and β2 microglobulin polypeptides. Thus, the MHC I expressed by the animals may be a chimeric human/non-human, e.g., human/rodent (e.g., human/mouse) MHC I polypeptide. A human portion of the chimeric MHC I polypeptide may be derived from a human HLA class I protein selected from the group consisting of HLA-A, HLA-B, and HLA-C, e.g., HLA-A2, HLA-B27, HLA-B7, HLA-Cw6, or any other HLA class I molecule present in a human population. In the embodiment, wherein the animal is a mouse, a non-human (i.e., a mouse) portion of the chimeric MHC I polypeptide may be derived from a mouse MHC I protein selected from H-2D, H-2K and H-2L.

In one embodiment, the non-human animal (e.g., rodent, e.g., mouse) of the invention further comprises a nucleotide sequence encoding a human or humanized MHC II protein, such that the chimeric CD4 protein expressed on the surface of a T cell of the animal is capable of interacting with a human or humanized MHC II expressed on a surface of a second cell, e.g., an antigen presenting cell. In one embodiment, the MHC II protein comprises an extracellular domain of a human MHC II α polypeptide and an extracellular domain of a human MHC II β polypeptide. Exemplary genetically modified animals expressing a human or humanized MHC II polypeptide are described in U.S. Pat. No. 8,847,005, issued Sep. 30, 2014, and U.S. Patent Publication No. 20130185820, incorporated herein by reference in their entireties. Thus, in one embodiment, the animal comprising chimeric CD4 protein described herein may further comprise a humanized MHC II protein, wherein the humanized MHC II protein comprises: (1) a humanized MHC II α polypeptide comprising a human MHC II α extracellular domain and transmembrane and cytoplasmic domains of an endogenous, e.g., mouse, MHC II, wherein the human MHC II α extracellular domain comprises α1 and α2 domains of a human MHC II α and (2) a humanized MHC II β polypeptide comprising a human MHC II β extracellular domain and transmembrane and cytoplasmic domains of an endogenous, e.g., mouse, MHC II, wherein the human MHC II β extracellular domain comprises β1 and β2 domains of a human MHC II β. In one aspect, both humanized MHC II α and β polypeptides are encoded by nucleic acid sequences located at endogenous MHC II α and β loci, respectively; in one aspect, the animal does not express functional endogenous MHC II α and β polypeptides. Thus, the MHC II expressed by the animals may be a chimeric human/non-human, e.g., human/rodent (e.g., human/mouse) MHC II protein. A human portion of the chimeric MHC II protein may be derived from a human HLA class II protein selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP, e.g., HLA-DR4, HLA-DR2, HLA-DQ2.5, HLA-DQ8, or any other HLA class II molecule present in a human population. In the embodiment, wherein the animal is a mouse, a non-human (i.e., a mouse) portion of the chimeric MHC II polypeptide may be derived from a mouse MHC II protein selected from H-2E and H-2A.

Various other embodiments of a genetically modified non-human animal, e.g. rodent, e.g., rat or mouse, would be evident to one skilled in the art from the present disclosure and from the disclosure of U.S. Patent Publication Nos. 20130111617, 20130185819 and 20130185820, and U.S. Pat. No. 8,847,005, incorporated herein by reference.

In various embodiments, the genetically modified non-human animals described herein make cells, e.g., APCs, with human or humanized MHC I and II on the cell surface and, as a result, present peptides as epitopes for T cells in a human-like manner, because substantially all of the components of the complex are human or humanized. The genetically modified non-human animals of the invention can be used to study the function of a human immune system in the humanized animal; for identification of antigens and antigen epitopes that elicit immune response (e.g., T cell epitopes, e.g., unique human cancer epitopes), e.g., for use in vaccine development; for evaluation of vaccine candidates and other vaccine strategies; for studying human autoimmunity; for studying human infectious diseases; and otherwise for devising better therapeutic strategies based on human MHC expression.

Non-Human Animals, Tissues and Cells

The genetically modified non-human animal of the invention may be selected from a group consisting of a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey). For the non-human animals where suitable genetically modifiable ES cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification. Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo.

In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal, e.g., of the superfamily Dipodoidea or Muroidea. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the superfamily Muroidea. In one embodiment, the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, white-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rates, bamboo rats, and zokors). In a specific embodiment, the genetically modified rodent is selected from a true mouse or rat (family Muridae), a gerbil, a spiny mouse, and a crested rat. In one embodiment, the genetically modified mouse is from a member of the family Muridae. In one embodiment, the animal is a rodent. In a specific embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a mouse.

In a specific embodiment, the non-human animal is a rodent that is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In another embodiment, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/Svlm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature for strain 129 mice, Mammalian Genome 10:836, see also, Auerbach et al (2000) Establishment and Chimera Analysis of 129/SvEv- and C57BL/6-Derived Mouse Embryonic Stem Cell Lines). In an embodiment, the genetically modified mouse is a mix of an aforementioned 129 strain and an aforementioned C57BL/6 strain. In another specific embodiment, the mouse is a mix of aforementioned 129 strains, or a mix of aforementioned BL/6 strains. In a specific embodiment, the 129 strain of the mix is a 129S6 (129/SvEvTac) strain. In another embodiment, the mouse is a BALB strain, e.g., BALB/c strain. In yet another embodiment, the mouse is a mix of a BALB strain and another aforementioned strain. Non-human animals as provided herein may be a mouse derived from any combination of the aforementioned strains.

In one embodiment, the non-human animal is a rat. In one embodiment, the rat is selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment, the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

Thus, in one embodiment of the invention, a genetically modified mouse is provided, wherein the mouse comprises, e.g., in its genome, e.g., in its germline genome, (a) a first nucleotide sequence encoding a first chimeric human/murine T cell co-receptor polypeptide (e.g., CD4), a second nucleotide sequence encoding a second chimeric human/murine T cell co-receptor polypeptide (e.g., CD8α), and/or a third nucleotide sequence encoding a third chimeric human/murine T cell co-receptor polypeptide (e.g., CD8β), wherein a murine portion of each chimeric T cell co-receptor polypeptide comprises at least transmembrane and cytoplasmic domains of a murine T cell co-receptor, wherein a human portion of each chimeric polypeptide comprises an extracellular portion (or part thereof, e.g., one or more extracellular domains) of a human T cell co-receptor, and wherein the mouse expresses the first, second and/or third chimeric T cell co-receptor polypeptide; (b) an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a murine TCRα constant gene sequence and/or an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a murine TCRβ constant gene sequence; and optionally, (c) a first nucleic acid sequence encoding a first chimeric human/murine MHC polypeptide (e.g., MHC II α), a second nucleic acid sequence encoding a second chimeric human/murine MHC polypeptide (e.g., MHC II β) and/or a third nucleic acid sequence encoding a third chimeric human/murine MHC polypeptide (e.g., MHC I) and a β2 microglobulin locus encoding a human or humanized β2 microglobulin, wherein a human portion of each chimeric MHC polypeptide comprises an extracellular domain of a human MHC polypeptide that associates with the first, second and/or third chimeric T cell co-receptor polypeptide (e.g., wherein a human portion of a chimeric MHC II complex (e.g., humanized MHC II α and β polypeptides) associates with the chimeric CD4 polypeptide and/or a human portion of the chimeric MHC I polypeptide (or MHC I complex, e.g., humanized MHC Iα and human(ized) β2 microglobulin) associates with the chimeric CD8 co-receptor (e.g., humanized CD8 α and β polypeptides).

A genetically modified mouse is provided herein comprising in its genome, e.g., at its endogenous CD4 locus, a nucleotide sequence encoding a chimeric human/mouse CD4 polypeptide, wherein a mouse portion of the chimeric polypeptide comprises at least transmembrane and cytoplasmic domains of a mouse CD4 polypeptide, and wherein the mouse expresses a chimeric human/mouse CD4. In one embodiment, a human portion of the chimeric polypeptide comprises at least all or substantially all of the extracellular domain of a human CD4 polypeptide. In one embodiment, a human portion of the chimeric polypeptide comprises at least all or substantially all of the D1 domain of a human CD4 protein. In one embodiment, a human portion of the chimeric polypeptide comprises at least all or substantially all of D1-D2 domains of a human CD4 protein, e.g., at least all or substantially all of D1-D3 domains of a human CD4 protein, e.g., all or substantially all of D1-D4 domains of a human CD4 protein. Thus, in one embodiment, the mouse comprises at the endogenous CD4 locus a nucleotide sequence comprising at least all or substantially all of exons 4, 5, and 6 of the human CD4 gene, e.g., the sequence of exon 3 of the human CD4 gene encoding a portion of the D1 domain of human CD4 and exons 4-6 of the human CD4 gene. In one embodiment, the mouse comprises at the endogenous CD4 locus a chimeric human/mouse CD4 that comprises a human CD4 sequence that is responsible for interacting with MHC II and/or extracellular portion of a T cell receptor. In another embodiment, the mouse comprises at the endogenous CD4 locus a chimeric human/mouse CD4 that comprises a human CD4 sequence that is responsible for interacting with MHC II and/or variable domain of a T cell receptor. In one embodiment, the nucleotide sequence comprises the sequence encoding mouse CD4 signal peptide. In one embodiment, the mouse comprises a replacement of the nucleotide sequence encoding a mouse CD4 extracellular domain with a nucleotide sequence encoding a human CD4 extracellular domain. In another embodiment, the mouse comprises a replacement of the nucleotide sequence encoding at least all or substantially all of mouse CD4 D1 domain, e.g., a nucleotide sequence encoding at least all or substantially all of mouse CD4 D1-D2 domains, e.g., a nucleotide sequence encoding at least all or substantially all of mouse CD4 D1-D3 domains, with human nucleotide sequence encoding the same. In one embodiment, the domains of chimeric CD4 polypeptide are encoded by a nucleotide sequence that is schematically represented in FIG. 5A.

In one embodiment, the mouse does not express a functional endogenous mouse CD4 from it endogenous mouse CD4 locus. In one embodiment, the mouse described herein comprises the chimeric human/mouse CD4 nucleotide sequence in the germline of the mouse.

In one embodiment, the mouse retains any endogenous sequences that have not been humanized, e.g., in the embodiment wherein the mouse comprises a replacement of the nucleotide sequence encoding all or substantially all of D1-D3 domains, the mouse retains endogenous nucleotide sequence encoding mouse CD4 D4 domain as well a nucleotide sequence encoding transmembrane and cytoplasmic domains of mouse CD4.

In one aspect, the mouse expressing chimeric human/mouse CD4 protein retains mouse CD4 promoter and regulatory sequences, e.g., the nucleotide sequence in the mouse encoding chimeric human/mouse CD4 is operably linked to endogenous mouse CD4 promoter and regulatory sequences. In one aspect, these mouse regulatory sequences retained in the genetically engineered animal of the invention include the sequences that regulate expression of the chimeric protein at proper stages during T cell development. Thus, in one aspect, the mouse does not express chimeric CD4 on B cells or mature CD8+ T cells. In one aspect, the mouse also does not express chimeric CD4 on any cell type, e.g., any immune cell type, that normally does not express endogenous CD4.

A genetically modified mouse disclosed herein may comprise in its genome, e.g., at its endogenous CD8 locus, a first nucleotide sequence encoding a chimeric human/mouse CD8α polypeptide and a second nucleotide sequence encoding a chimeric human/mouse CD8β polypeptide. In one embodiment, the first nucleotide sequence comprises a sequence that encodes all or substantially all of an extracellular portion of a human CD8α polypeptide and at least transmembrane and cytoplasmic domains of a mouse CD8α polypeptide, and the second nucleotide sequence comprises a sequence that encodes all or substantially all of an extracellular portion of a human CD8β polypeptide and at least transmembrane and cytoplasmic domains of a mouse CD8β polypeptide, and wherein the mouse expresses a functional chimeric human/mouse CD8 protein. In one embodiment, the first nucleotide sequence comprises a sequence that encodes at least the immunoglobulin V-like domain of the human CD8α polypeptide and the remaining sequences of a mouse CD8α polypeptide, and the second nucleotide sequence comprises a sequence that encodes at least the immunoglobulin V-like domain of the human CD8β polypeptide and the remaining sequences of a mouse CD8β polypeptide. In one embodiment, first nucleotide sequence comprises at least the MHC I-binding domain of a human CD8α polypeptide. In one embodiment, the first and the second nucleotide sequences comprise at least the exons that encode the extracellular portion of a human CD8α polypeptide and/or CD8β polypeptide, respectively. In one embodiment, the extracellular portion of a human CD8α polypeptide and/or CD8β polypeptide is a region encompassing the portion of the human CD8α polypeptide and/or CD8β polypeptide that is not transmembrane or cytoplasmic domain. In one embodiment, the domains of a chimeric CD8α polypeptide are encoded by a nucleotide sequence that is schematically represented in FIG. 5B. In one embodiment, the domains of a chimeric CD8β polypeptide are encoded by a nucleotide sequence that is schematically represented in FIG. 5B. In one embodiment, the nucleotide sequence encoding the chimeric human/mouse CD8α polypeptide and/or CD8β polypeptide comprises the sequence encoding a mouse CD8α and/or CD8β signal peptide, respectively. Alternatively, the nucleotide sequence may comprise the sequence encoding a human CD8α and/or CD8β signal sequence. In one embodiment, the mouse comprises a replacement of a nucleotide sequence encoding all or substantially all of the mouse CD8α and/or CD8β extracellular domain with a nucleotide sequence encoding all or substantially all of the human CD8α and/or CD8β extracellular domain, respectively.

In one embodiment, the mouse does not express a functional endogenous mouse CD8α and/or CD8β polypeptide from its endogenous CD8 locus. In one embodiment, the mouse as described herein comprises the chimeric human/mouse CD8 sequence in its germline.

In one aspect, the mouse expressing chimeric human/mouse CD8α and/or CD8β polypeptide retains mouse CD8α and/or CD8β promoter and regulatory sequences, e.g., the nucleotide sequence in the mouse encoding chimeric human/mouse CD8 is operably linked to endogenous mouse CD8 promoter and regulatory sequences. In one aspect, these regulatory sequences retained in the mouse include the sequences regulating CD8 protein expression at proper stages of T cell development. In one aspect, the genetically modified mouse does not express chimeric CD8 on B cells or mature CD4+ T cells, or any cell, e.g., immune cell, that does not normally express endogenous CD8.

The invention also provides a genetically modified mouse comprising in its genome an unrearranged human or humanized TCR variable gene locus, e.g., TCRα, TCRβ, TCRδ, and/or TCRγ variable gene locus. In some embodiments, the unrearranged human or humanized TCR variable gene locus replaces endogenous mouse TCR variable gene locus. In other embodiments, unrearranged human or humanized TCR variable gene locus is at a site in the genome other than the corresponding endogenous mouse TCR locus. In some embodiments, human or humanized unrearranged TCR variable gene locus is operably linked to mouse TCR constant region.

In one embodiment, a genetically modified mouse is provided, wherein the mouse comprises in its genome an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and least one human Jα segment, operably linked to a mouse TCRα constant gene sequence, and an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a mouse TCRβ constant gene sequence. In one specific embodiment, the mouse comprises in its genome an unrearranged TCRα variable gene locus comprising a complete repertoire of human Vα segments and a complete repertoire of human Jα segments operably linked to a mouse TCRα constant gene sequence, and an unrearranged TCRβ variable gene locus comprising a complete repertoire of human Vβ segments, a complete repertoire of human Dβ segments, and a complete repertoire of human Jβ segments operably linked to a mouse TCRβ constant gene sequence.

In some embodiments, the unrearranged TCRα variable gene locus comprising human TCRα variable region segments replaces endogenous mouse TCRα variable gene locus, and the unrearranged TCRβ variable gene locus comprising human TCRβ variable region segments replaces the endogenous mouse TCRβ variable gene locus. In some embodiments, the endogenous mouse Vα and Jα segments are incapable of rearranging to form a rearranged Vα/Jα sequence, and the endogenous mouse Vβ, Dβ, and Jβ segments are incapable of rearranging to form a rearranged Vβ/Dβ/Jβ sequence. In some embodiments, the human Vα and Jα segments rearrange to form a rearranged human Vα/Jα sequence, and the human Vβ, Dβ, and Jβ segments rearrange to form a rearranged human Vβ/Dβ/Jβ sequence.

The invention also relates to a genetically modified mouse that comprises in its genome a nucleic acid sequence encoding a chimeric MHC polypeptide, wherein the human portion of the chimeric MHC polypeptide associates with a human extracellular domain of a chimeric T cell co-receptor as disclosed herein. Genetically modified mice as disclosed herein may comprise a first nucleic acid sequence encoding a chimeric human/mouse MHC I, a second nucleic acid sequence encoding a chimeric human/mouse MHC II α, and/or a third nucleic acid sequence encoding a chimeric human/mouse MHC II β polypeptides. A human portion of the chimeric MHC I, MHC II α, and/or MHC II β may comprise an extracellular domain of a human MHC I, MHC II α, and MHC II β, respectively. In one embodiment, the mouse expresses functional chimeric human/mouse MHC I, MHC II α, and MHC II β polypeptides from its endogenous mouse MHC locus. In one embodiment, the mouse does not express functional mouse MHC polypeptides, e.g., functional mouse MHC I, MHC II α, and MHC II β polypeptides, from its endogenous mouse MHC locus. In other embodiments, the only MHC I and MHC II expressed by the mouse on a cell surface are chimeric MHC I and II.

In one embodiment, a human portion of the chimeric human/mouse MHC I polypeptide comprises a peptide binding domain or an extracellular domain of a human MHC I (e.g., human HLA-A, e.g., human HLA-A2, e.g., human HLA-A2.1). In some embodiments, the mouse does not express a peptide binding or an extracellular domain of an endogenous mouse MHC I polypeptide from its endogenous mouse MHC I locus. The peptide binding domain of the human MHC I may comprise α1 and α2 domains. Alternatively, the peptide binding domain of the human MHC I may comprise α1, α2, and α3 domains. In one aspect, the extracellular domain of the human MHC I comprises an extracellular domain of a human MHC I α chain. In one embodiment, the endogenous mouse MHC I locus is an H-2K (e.g., H-2Kb) locus, and the mouse portion of the chimeric MHC I polypeptide comprises transmembrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb) polypeptide. Thus, in one embodiment, the mouse of the invention comprises at its endogenous mouse MHC I locus a nucleic acid sequence encoding a chimeric human/mouse MHC I, wherein a human portion of the chimeric polypeptide comprises an extracellular domain of a human HLA-A2 (e.g., HLA-A2.1) polypeptide and a mouse portion comprises transmembrane and cytoplasmic domains of a mouse H-2K (e.g., H-2Kb) polypeptide, and a mouse expresses a chimeric human/mouse HLA-A2/H-2K protein. In other embodiment, the mouse portion of the chimeric MHC I polypeptide may be derived from other mouse MHC I, e.g., H-2D, H-2L, etc.; and the human portion of the chimeric MHC I polypeptide may be derived from other human MHC I, e.g., HLA-B, HLA-C, etc. In one aspect, the mouse does not express a functional endogenous H-2K polypeptide from its endogenous mouse H-2K locus. In one embodiment, the mouse does not express functional endogenous MHC polypeptides from its H-2D locus. In some embodiments, the mouse is engineered to lack all or a portion of an endogenous H-2D locus. In other embodiments, the only MHC I polypeptides expressed by the mouse on a cell surface are chimeric human/mouse MHC I polypeptides.

In one embodiment, a human portion of the chimeric human/mouse MHC II α polypeptide comprises a human MHC II α peptide binding or extracellular domain and a human portion of the chimeric human/mouse MHC II β polypeptide comprises a human MHC II β peptide binding or extracellular domain. In some embodiments, the mouse does not express a peptide binding or an extracellular domain of endogenous mouse a and/or polypeptide from an endogenous mouse locus (e.g., H-2A and/or H-2E locus). In some embodiments, the mouse comprises a genome that lacks a gene that encodes a functional MHC class II molecule comprising an H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, H-2Ea, and a combination thereof. In some embodiments, the only MHC II polypeptides expressed by the mouse on a cell surface are chimeric human/mouse MHC II polypeptides. The peptide-binding domain of the human MHC II α polypeptide may comprise α1 domain and the peptide-binding domain of the human MHC II β polypeptide may comprise a β1 domain; thus, the peptide-binding domain of the chimeric MHC II complex may comprise human α1 and β1 domains. The extracellular domain of the human MHC II α polypeptide may comprise α1 and α2 domains and the extracellular domain of the human MHC II β polypeptide may comprise β1 and β2 domains; thus, the extracellular domain of the chimeric MHC II complex may comprise human α1, α2, β1 and β2 domains. In one embodiment, the mouse portion of the chimeric MHC II complex comprises transmembrane and cytosolic domains of mouse MHC II, e.g. mouse H-2E (e.g., transmembrane and cytosolic domains of mouse H-2E α and β chains). Thus, in one embodiment, the mouse of the invention comprises at its endogenous mouse MHC II locus a nucleic acid sequence encoding a chimeric human/mouse MHC II α, wherein a human portion of the chimeric MHC II α polypeptide comprises an extracellular domain derived from an α chain of a human MHC II (e.g., α chain of HLA-DR2) and a mouse portion comprises transmembrane and cytoplasmic domains derived from an α chain of a mouse MHC II (e.g., H-2E); and a mouse comprises at its endogenous mouse MHC II locus a nucleic acid sequence encoding a chimeric human/mouse MHC II β, wherein a human portion of the chimeric MHC II β polypeptide comprises an extracellular domain derived from a β chain of a human MHC II (e.g., β chain of HLA-DR2) and a mouse portion comprises transmembrane and cytoplasmic domains derived from a β chain of a mouse MHC II (e.g., H-2E); e.g., wherein the mouse expresses a chimeric human/mouse HLA-DR2/H-2E protein. In other embodiment, the mouse portion of the chimeric MHC II protein may be derived from other mouse MHC II, e.g., H-2A, etc.; and the human portion of the chimeric MHC II protein may be derived from other human MHC II, e.g., HLA-DQ, etc. In one aspect, the mouse does not express functional endogenous H-2A and H-2E polypeptides from their endogenous mouse loci (e.g., the mouse does not express H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea polypeptides). In some embodiments, the mouse lacks expression of any endogenous MHC I or MHC II molecule on a cell surface.

In addition to at least one humanized T cell co-receptor, at least one humanized MHC that associates with the humanized T cell co-receptor, and optionally, a humanized TCR, a genetically modified non-human animal as described herein may also express a human or humanized β2 microglobulin. In various aspects, the human or humanized β2 microglobulin expressed by a genetically modified non-human animal, or cells, embryos, or tissues derived from a non-human animal, preserves all the functional aspects of the endogenous and/or human β2 microglobulin. For example, it is preferred that the human or humanized β2 microglobulin binds the α chain of MHC I polypeptide (e.g., endogenous non-human or human MHC I polypeptide). The human or humanized β2 microglobulin polypeptide may bind, recruit or otherwise associate with any other molecules, e.g., receptor, anchor or signaling molecules that associate with endogenous non-human and/or human β2 microglobulin (e.g., HFE, etc.).

In addition to genetically modified animals (e.g., rodents, e.g., mice or rats), also provided is a tissue or cell, wherein the tissue or cell is derived from a non-human animal as described herein, and comprises a heterologous β2 microglobulin gene or β2 microglobulin sequence, i.e., nucleotide and/or amino acid sequence. In one embodiment, the heterologous β2 microglobulin gene or β2 microglobulin sequence is a human or humanized β2 microglobulin gene or human or humanized β2 microglobulin sequence. Preferably, the cell is a nucleated cell. The cell may be any cell known to express MHC I complex, e.g., an antigen presenting cell. The human or humanized β2 microglobulin polypeptide expressed by said cell may interact with endogenous non-human MHC I (e.g., rodent MHC I), to form a functional MHC I complex. The resultant MHC I complex may be capable of interacting with a T cell, e.g., a cytotoxic T cell. Thus, also provided is an in vitro complex of a cell from a non-human animal as described herein and a T cell.

Also provided are non-human cells that comprise human or humanized β2 microglobulin gene or sequence, and an additional human or humanized sequence, e.g., chimeric MHC I polypeptide presently disclosed. In such an instance, the human or humanized β2 microglobulin polypeptide may interact with, e.g., a chimeric human/non-human MHC I polypeptide, and a functional MHC I complex may be formed. In some aspects, such complex is capable of interacting with a TCR on a T cell, e.g., a human or a non-human T cell. Thus, also provided is an in vitro complex of a cell from a non-human animal as described herein and a human or a non-human T cell.

Another aspect of the disclosure is a rodent embryo (e.g., a mouse or a rat embryo) comprising a heterologous β2 microglobulin gene or β2 microglobulin sequence as described herein. In one embodiment, the embryo comprises an ES donor cell that comprises the heterologous β2 microglobulin gene or β2 microglobulin sequence, and host embryo cells. The heterologous β2 microglobulin gene or β2 microglobulin sequence is a human or humanized β2 microglobulin gene or β2 microglobulin sequence.

This invention also encompasses a non-human cell comprising a chromosome or fragment thereof of a non-human animal as described herein (e.g., wherein the chromosome or fragment thereof comprises a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide). The non-human cell may comprise a nucleus of a non-human animal as described herein. In one embodiment, the non-human cell comprises the chromosome or fragment thereof as the result of a nuclear transfer.

In one aspect, a non-human induced pluripotent cell comprising a heterologous β2 microglobulin gene or β2 microglobulin sequence is provided. In one embodiment, the induced pluripotent cell is derived from a non-human animal as described herein. In one embodiment, the heterologous β2 microglobulin gene or β2 microglobulin sequence is a human or humanized gene or sequence.

In some embodiments of the invention, the mouse described herein expresses chimeric human/mouse MHC II only on professional antigen presenting cells, e.g., B cell, monocytes/macrophages, and/or dendritic cells of the mouse. In some embodiments, a mouse described herein elicits an immune response, e.g., a cellular immune response, to one or more human antigens. In some embodiments, a mouse described herein elicits a humanized T cell response to one or more human antigens.

In addition to a genetically engineered non-human animal, a non-human embryo (e.g., a rodent, e.g., a mouse or a rat embryo) is also provided, wherein the embryo comprises a donor ES cell that is derived from a non-human animal (e.g., a rodent, e.g., a mouse or a rat) as described herein. In one aspect, the embryo comprises an ES donor cell that comprises the chimeric CD4 gene, the chimeric CD8 (e.g., CD8α and/or CD8β) gene, a humanized MHC I (e.g., MHC I α) nucleic acid sequence, a humanized MHC II (e.g., MHC II α and/or MHC II β) nucleic acid sequence, an unrearranged humanized TCR (e.g., TCRα and/or TCRβ, or TCRδ, and/or TCRγ) locus and/or human or humanized β2 microglobulin gene sequence and host embryo cells.

Also provided is a tissue, wherein the tissue is derived from a non-human animal (e.g., a rodent, e.g., a mouse or a rat) as described herein, and expresses the chimeric CD4 protein, the chimeric CD8 protein (e.g., chimeric CD8α and/or CD8β protein), a humanized TCR polypeptide (e.g., TCRα and/or TCRβ, or TCRδ, and/or TCRγ polypeptide), a humanized MHC I polypeptide (e.g., MHC I α), a humanized MHC II polypeptide (e.g., MHC II α and/or MHC II β polypeptide) and/or a human or humanized β2 microglobulin.

In one aspect, a method for making a chimeric human/non-human CD4 molecule is provided, comprising expressing in a single cell a chimeric CD4 protein from a nucleotide construct as described herein. In one embodiment, the nucleotide construct is a viral vector; in a specific embodiment, the viral vector is a lentiviral vector. In one embodiment, the cell is selected from a CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).

In one aspect, a cell that expresses a chimeric CD4 protein is provided. In one embodiment, the cell comprises an expression vector comprising a chimeric CD4 sequence as described herein. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).

A chimeric CD4 molecule made by a non-human animal as described herein is also provided, wherein, in one embodiment, the chimeric CD4 molecule comprises an amino acid sequence of all or substantially all of an extracellular domain of a human CD4 protein, and at least transmembrane and cytoplasmic domains from a non-human CD4 protein, e.g., mouse CD4 protein. In another embodiment, a chimeric CD4 molecule made by a non-human animal as described herein is provided, wherein the chimeric CD4 molecule comprises an amino acid sequence of at least all or substantially all D1 domain of a human CD4, e.g., at least all or substantially all D1-D2 domains of a human CD4, e.g., at least all or substantially all D1-D3 domains of a human CD4, e.g., an amino acid sequence of human CD4 that is responsible for binding MHC II and/or extracellular domain of a TCR, e.g., an amino acid sequence of human CD4 that is responsible for binding MHC II and/or a variable domain of a TCR; and wherein the remainder of the protein (e.g., transmembrane domain, cytoplasmic domain, any portion of extracellular domain that has not been humanized) is derived from the endogenous non-human protein sequence. An exemplary chimeric human/non-human CD4 polypeptide comprises an amino acid sequence set forth in SEQ ID NO:78, and the human portion of the chimeric polypeptide spans about amino acids 27-319 of SEQ ID NO:78 (set forth separately in SEQ ID NO:79).

In one aspect, a method for making a chimeric human/non-human CD8 molecule (e.g., CD8α and/or CD8β) is provided, comprising expressing in a single cell a chimeric CD8 polypeptide(s) from a nucleotide construct(s) as described herein. In one embodiment, the nucleotide construct is a viral vector; in a specific embodiment, the viral vector is a lentiviral vector. In one embodiment, the cell is selected from a CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).

In one aspect, a cell that expresses a chimeric CD8 protein is provided. In one embodiment, the cell comprises an expression vector comprising a chimeric CD8 sequence(s) as described herein. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).

A chimeric CD8 molecule made by a non-human animal as described herein is also provided, wherein the chimeric CD8 molecule comprises all or substantially all of the extracellular domain from a human CD8 protein (e.g., CD8α and/or CD8β), and at least transmembrane and cytoplasmic domains from a non-human CD8 protein, e.g., mouse CD8 protein. Exemplary chimeric CD8α polypeptide is set forth in SEQ ID NO:88, and exemplary chimeric CD8β protein is set forth in SEQ ID NO:83.

A humanized TCR protein made by a non-human animal (e.g., rodent, e.g., mouse or rat) as described herein is also provided, wherein the humanized TCR protein comprises a human variable region and a non-human constant region. Thus, the humanized TCR protein comprises human complementary determining regions (i.e., human CDR1, 2, and 3) in its variable domain and a non-human constant region. Also provided are nucleic acids that encode the human TCR variable domains generated by a non-human animal described herein.

In addition, a non-human cell isolated from a non-human animal as described herein is provided. In one embodiment, the cell is an ES cell. In one embodiment, the cell is a T cell, e.g., a CD4+ T cell. In one embodiment, the cell is a helper T cell (T_(H) cell). In one embodiment, the T_(H) cell is an effector T_(H) cell, e.g., T_(H)1 cell or T_(H)2 cell. In one embodiment, the cell is CD8+ T cell. In one embodiment, the cell is a cytotoxic T cell. Also provided is a non-human cell that expresses a TCR protein comprising a human variable region and a non-human constant region. The TCR protein may comprise TCRα, TCRβ, or a combination thereof. In one embodiment, the cell is a T cell, e.g., a CD4+ or a CD8+ T cell. Additionally, non-human T cells as provided herein may express on its cell surface (a) a chimeric human/non-human T cell co-receptor, e.g., a chimeric CD4 polypeptide or a chimeric CD8 polypeptide, comprising a human T cell co-receptor extracellular domain operably linked to a non-human T cell co-receptor transmembrane and/or intracellular domain; and (b) a TCR protein comprising a human variable region and a non-human constant region.

In another embodiment, the cell is an antigen presenting cell. In one embodiment, the antigen presenting cell presents antigen on humanized MHC I molecules. In another embodiment, the antigen presenting cell is a professional antigen presenting cell, e.g., a B cell, a dendritic cell, and a macrophage. In another embodiment, the antigen presenting cell presents antigen on humanized MHC I and/or humanized MHC II molecules.

In one aspect, a cell that expresses a chimeric human/non-human MHC I and MHC II proteins (e.g., HLA-A2/H-2K and HLA-DR2/H-2E proteins) is provided. In one aspect, the cell is a mouse cell that does not express functional endogenous MHC polypeptides from its H-2D locus. In some embodiments, the cell is a mouse cell engineered to lack all or a portion of an endogenous H-2D locus. In some embodiments, the cell is a mouse cell that does not express any functional endogenous MHC I and MHC II polypeptide on its surface. In one embodiment, the cell comprises an expression vector comprising a chimeric MHC class I sequence and chimeric MHC class II sequence as described herein. In one embodiment, the cell is selected from CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).

A chimeric MHC II complex comprising an extracellular domain of HLA-DR2 described herein may be detected by anti-HLA-DR antibodies. Thus, a cell displaying chimeric human/non-human MHC II polypeptide may be detected and/or selected using anti-HLA-DR antibody. The chimeric MHC I complex comprising an extracellular domain of HLA-A2 described herein may be detected using anti-HLA-A, e.g., anti-HLA-A2 antibodies. Thus, a cell displaying a chimeric human/non-human MHC I polypeptide may be detected and/or selected using anti-HLA-A antibody. Antibodies that recognize other HLA alleles are commercially available or can be generated, and may be used for detection/selection.

Although the Examples that follow describe a genetically engineered animal whose genome comprises a replacement of a nucleic acid sequence encoding mouse H-2K, and H-2A and H-2E proteins with a nucleic acid sequence encoding a chimeric human/mouse HLA-A2/H-2K and HLA-DR2/H-2E protein, respectively, one skilled in the art would understand that a similar strategy may be used to introduce chimeras comprising other human MHC I and II genes (other HLA-A, HLA-B, and HLA-C; and other HLA-DR, HLA-DP and HLA-DQ genes). Such animals comprising multiple chimeric human/non-human (e.g., human/rodent, e.g., human/mouse) MHC I and MHC II genes at endogenous MHC loci are also provided. Examples of such chimeric MHC I and MHC II proteins are described in U.S. Publication Nos. 20130111617, 20130185819, 20130185820 and 20140245467 and U.S. Pat. No. 8,847,005, each of which are incorporated herein by reference.

Also provided is a non-human cell comprising a chromosome or fragment thereof of a non-human animal as described herein. In one embodiment, the non-human cell comprises a nucleus of a non-human animal as described herein. In one embodiment, the non-human cell comprises the chromosome or fragment thereof as the result of a nuclear transfer.

In one aspect, a non-human induced pluripotent cell comprising a gene encoding a chimeric CD4 polypeptide, a gene encoding a chimeric CD8 polypeptide (e.g., CD8α and/or CD8β polypeptide), a gene encoding a humanized MHC I polypeptide (e.g., MHC I α and/or β2 microglobulin), a gene encoding a humanized MHC II polypeptide (e.g., MHC II α and/or MHC II β) and/or an unrearranged humanized TCR locus encoding a humanized TCRα and/or TCRβ polypeptide as described herein is provided. In one embodiment, the induced pluripotent cell is derived from a non-human animal as described herein.

In one aspect, a hybridoma or quadroma is provided, derived from a cell of a non-human animal as described herein. In one embodiment, the non-human animal is a mouse or rat.

Making Genetically Modified Non-Human Animals that Mount Substantially Humanized T Cell Immune Responses

Also provided is a method for making a genetically engineered non-human animal (e.g., a genetically engineered rodent, e.g., a mouse or rat) described herein. Generally, the methods comprise (a) introducing into the genome of the non-human animal a first nucleotide sequence encoding a chimeric human/non-human T cell co-receptor polypeptide, a second nucleotide sequence encoding a second chimeric human/non-human T cell co-receptor polypeptide, and/or a third nucleotide sequence encoding a third chimeric human/non-human T cell co-receptor polypeptide, wherein a non-human portion of each chimeric T cell co-receptor polypeptide comprises at least transmembrane and cytoplasmic domains of a non-human T cell co-receptor, and wherein a human portion of each chimeric polypeptide comprises an extracellular portion (or part thereof) of a human T cell co-receptor; (b) inserting into the genome of the non-human animal an unrearranged T cell receptor (TCR) α variable gene locus comprising at least one human Vα segment and at least one human Jα segment, operably linked to a non-human TCRα constant gene sequence and/or an unrearranged TCRβ variable gene locus comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment, operably linked to a non-human TCRβ constant gene sequence; and optionally (c) placing into the genome a first nucleic acid sequence encoding a first chimeric human/non-human MHC polypeptide, a second nucleic acid sequence encoding a second chimeric human/non-human MHC polypeptide and/or a third nucleic acid sequence encoding a third chimeric human/non-human MHC polypeptide and/or (d) adding into the genome of the non-human animal a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide. In some embodiments, the steps of introducing, inserting and/or placing comprise targeting sequences encoding the extracellular domain(s) of the T cell co-receptor, the variable domain(s) of the TCR, the extracellular domain(s) of the MHC polypeptide, or a portion of the β2 microglobulin and replacing them with sequences encoding human T cell co-receptor extracellular domain(s), human TCR variable domains, human MHC extracellular domain(s), and/or a human portion of the β2 microglobulin, respectively.

In other embodiments, introducing, inserting, placing and/or adding may comprise breeding, e.g., mating, animals of the same species. In other embodiments, introducing, inserting, placing and/or adding comprises sequential homologous recombination in ES cells. In some embodiments, the ES cells are derived from non-human animals genetically modified to comprise one or more, but not all, of the genetic modifications desired, and homologous recombination in such ES cells completes the genetic modification. In other embodiments, introducing, inserting, placing and/or adding may comprise a combination of breeding and homologous recombination in ES cells, e.g., breeding an animal to another (or more) animal of the same species, wherein some or all of the animals may be generated from ES cells genetically modified via a single homologous recombination or sequential homologous recombination events, and wherein some ES cell may be isolated from a non-human animal comprising one or more of the genetic modifications disclosed herein.

In some embodiments, the method utilizes a targeting construct made using VELOCIGENE® technology, introducing the construct into ES cells, and introducing targeted ES cell clones into a mouse embryo using VELOCIMOUSE® technology, as described in the Examples. Targeting construct may comprise 5′ and/or 3′ homology arms that target the endogenous sequence to be replaced, an insert sequence (that replaces the endogenous sequence) and one or more selection cassettes. A selection cassette is a nucleotide sequence inserted into a targeting construct to facilitate selection of cells (e.g., ES cells) that have integrated the construct of interest. A number of suitable selection cassettes are known in the art. Commonly, a selection cassette enables positive selection in the presence of a particular antibiotic (e.g., Neo, Hyg, Pur, CM, SPEC, etc.). In addition, a selection cassette may be flanked by recombination sites, which allow deletion of the selection cassette upon treatment with recombinase enzymes. Commonly used recombination sites are loxP and Frt, recognized by Cre and Flp enzymes, respectively, but others are known in the art. A selection cassette may be located anywhere in the construct outside the coding region. In one embodiment, the selection cassette is located at the 5′ end the human DNA fragment. In another embodiment, the selection cassette is located at the 3′ end of the human DNA fragment. In another embodiment, the selection cassette is located within the human DNA fragment. In another embodiment, the selection cassette is located within an intron of the human DNA fragment. In another embodiment, the selection cassette is located at the junction of the human and mouse DNA fragment.

In one embodiment, the method for making a genetically engineered non-human animal results in the animal that comprises at an endogenous CD4 locus a nucleotide sequence encoding a chimeric human/non-human CD4 polypeptide. In one embodiment, the invention comprises a method of modifying a CD4 locus of a non-human animal to express a chimeric human/non-human CD4 polypeptide described herein. In one embodiment, the invention provides a method of modifying a CD4 locus of a mouse to express a chimeric human/mouse CD4 polypeptide comprising introducing, e.g., replacing at an endogenous CD4 locus of a non-human animal, e.g., a mouse, a nucleotide sequence encoding an endogenous non-human CD4 polypeptide with a nucleotide sequence encoding a chimeric human/mouse CD4 polypeptide. In one aspect of the method, the chimeric human/mouse CD4 polypeptide comprises all or substantially all of an extracellular domain of a human CD4 polypeptide and at least transmembrane and cytoplasmic domains of an endogenous mouse CD4 polypeptide. In another aspect of the method, the chimeric human/mouse CD4 polypeptide comprises all or substantially all of D1-D2 domains of a human CD4 polypeptide. In yet another embodiment, the chimeric human/mouse CD4 polypeptide comprises all or substantially all of D1-D3 domains of a human CD4 polypeptide. In yet another embodiment, the chimeric human/mouse CD4 polypeptide comprises all or substantially all of amino acid sequence of human CD4 that is responsible for interacting with MHC II and/or an extracellular domain of a T cell receptor. In yet another embodiment, the chimeric human/mouse CD4 polypeptide comprises all or substantially all of amino acid sequence of human CD4 that is responsible for interacting with MHC II and/or a variable domain of a T cell receptor.

Thus, a nucleotide construct for generating genetically modified animals comprising chimeric human/non-human CD4 is provided. In one aspect, the nucleotide sequence comprises 5′ and 3′ homology arms, a DNA fragment comprising human CD4 gene sequence (e.g., human CD4 extracellular domain gene sequence, e.g., gene sequence of all or substantially all of domains D1-D2 of human CD4, e.g., gene sequence of all or substantially all of domains D1-D3 and/or D2-D3 of human CD4, e.g., gene sequence of all or substantially all of domains D1-D4 of human CD4), and a selection cassette flanked by recombination sites. In one embodiment, human CD4 gene sequence is a genomic sequence that comprises introns and exons of human CD4. In one embodiment, homology arms are homologous to non-human (e.g., mouse) CD4 genomic sequence. An exemplary construct of the invention is depicted in FIG. 5A.

In some embodiments, the method results in an animal that comprises at an endogenous CD8 locus a nucleotide sequence(s) encoding a chimeric human/non-human CD8α and/or CD8β polypeptide. In one embodiment, the invention provides a method of modifying a CD8 locus of a non-human animal to express a chimeric human/non-human CD8 polypeptide described herein. In one aspect, provided is a method of modifying a CD8 locus of a mouse to express a chimeric human/mouse CD8 polypeptide comprising introducing, e.g., replacing, at an endogenous CD8 locus of a non-human animal, e.g., a mouse, a nucleotide sequence encoding an endogenous non-human CD8 polypeptide with a nucleotide sequence encoding a chimeric human/mouse CD8 polypeptide. The CD8 polypeptide may be selected from CD8α, CD8β, and combination thereof. In one aspect, the chimeric polypeptide comprises all or substantially all of an extracellular domain of a human CD8 polypeptide and at least transmembrane and cytoplasmic domains of an endogenous mouse CD8 polypeptide.

Thus, a nucleotide construct for generating genetically modified animals comprising human/non-human CD8 is also provided. In one aspect, the sequence of the nucleotide construct comprises 5′ and 3′ homology arms, a DNA fragment comprising human CD8α or CD8β sequence, and a selection cassette flanked by recombination sites. In some embodiments, the human sequence comprises introns and exons of human CD8α or CD8β, e.g., exons encoding the extracellular domain of the human CD8α or CD8β, respectively. In one embodiment, homology arms are homologous to non-human CD8α or CD8β sequence. Exemplary constructs for CD8α and CD8β are depicted in FIG. 5B.

Because of close chromosomal localization of the genes encoding CD8α and CD8β, sequential targeting of the two genes improves the chances of successful humanization. In one embodiment, the targeting strategy comprises introducing chimeric CD8β construct described herein into ES cells, generating a mouse from the targeted ES cells, deriving genetically modified ES cells from said mouse, and introducing chimeric CD8α construct described herein into said genetically modified ES cells. In another embodiment, the targeting strategy comprises introducing a chimeric CD8β construct described herein into ES cells, selecting the cells that have incorporated the chimeric CD8β construct, introducing a chimeric CD8α construct described herein into ES cells that have incorporated and are harboring the chimeric CD8β construct, and selecting the cells that have incorporated both chimeric CD8β and CD8α. In one aspect of this embodiment, the steps of selecting are performed utilizing different selection markers. In alternative embodiments, CD8α humanization can be accomplished first. Upon completion of gene targeting, ES cells of genetically modified non-human animals can be screened to confirm successful incorporation of exogenous nucleotide sequence of interest or expression of exogenous polypeptide by a variety of methods known in the art (e.g., modification of allele assay described in Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-659).

In some embodiments, the method for making a genetically modified non-human animal results in the animal whose genome comprises a humanized unrearranged TCR locus (e.g., a humanized unrearranged TCRα, TCRβ, TCRδ, and/or TCRγ locus). In one embodiment, a method for making a genetically modified non-human animal (e.g., rodent, e.g., mouse or rat) that expresses a T cell receptor comprising a human variable region and a non-human (e.g., rodent, e.g., mouse or rat) constant region on a surface of a T cell is provided, wherein the method comprises inserting, e.g., replacing, in a first non-human animal an endogenous non-human TCRα variable gene locus with an unrearranged humanized TCRα variable gene locus comprising at least one human Vα segment and at least one human Jα segment, wherein the humanized TCRα variable gene locus is operably linked to endogenous TCRα constant region; inserting, e.g., replacing in a second non-human animal an endogenous non-human TCRβ variable gene locus with an unrearranged humanized TCRβ variable gene locus comprising at least one human Vβ segment, one human Dβ segment, and one human Jβ segment, wherein the humanized TCRβ variable gene locus is operably linked to endogenous TCRβ constant region; and breeding the first and the second non-human animal to obtain a non-human animal that expresses a T cell receptor comprising a human variable region and a non-human constant region. In other embodiments, the invention provides methods of making a genetically modified non-human animal whose genome comprises a humanized unrearranged TCRα locus, or a non-human animal whose genome comprises a humanized unrearranged TCRβ locus. In various embodiments, the replacements are made at the endogenous loci. In various embodiments, the method comprises progressive humanization strategy, wherein a construct comprising additional variable region segments is introduced into ES cells at each subsequent step of humanization, ultimately resulting in a mouse comprising a complete repertoire of human variable region segments and fully human TCRBDJ1 and TCRBDJ2 clusters (see, e.g., FIGS. 4A and 4B). Some method embodiments described herein comprise (1) replacing an endogenous non-human (e.g., mouse) tcrbdj1 sequence with a nucleic acid sequence comprising a human TRBD1 and human TRBJ1-1 to TRBJ1-6 gene segments and non-human (e.g., mouse) tcrbdj1 non-coding sequences (including non-coding recombination signal sequences (RSSs) and other non-intergenic sequences), where the human TRBD1 and human TRBJ1-1 to TRBJ1-6 gene segments flank the same non-human (e.g., mouse) tcrbdj1 TCR non-coding sequences as are normally flanked by non-human (e.g., mouse) Trbd1 and non-human (e.g., mouse) Trbj1-1 to Trbj1-6 gene segments and/or (2) replacing an endogenous non-human (e.g., mouse) tcrbdj2 sequence with a nucleic acid sequence comprising a human TRBD2 and human TRBJ2-1 to TRBJ2-7 gene segments and mouse tcrbdj2 non-coding sequences, where the human TRBD2 and human TRBJ2-1 to TRBJ2-7 gene segments flank the same mouse Tcrbdj2 non-coding sequences as are normally flanked by the mouse Trbd2 and mouse Trbj2-1 to Trbj2-7 gene segments. In some embodiments, such replacements result in operable linkage of the nucleic acid sequence that comprises a human TRBD1 and human TRBJ1-1 to TRBJ1-6 gene segments and non-human (e.g., mouse) tcrbdj1 non-coding sequences (including non-coding recombination signal sequences (RSSs) and other non-intergenic sequences) to a non-human (e.g., mouse) tcrbc1 constant region sequence and/or the operable linkage of the sequence that comprises a human TRBD2 and human TRBJ2-1 to TRBJ2-7 gene segments and mouse TCRBDJ2 non-coding sequences (including non-coding recombination signal sequences (RSSs) and other non-intergenic sequences) to a non-human (e.g., mouse) tcrbc2 constant region sequence, respectively (See, FIG. 4C). In such embodiments, resulting mice may comprise one to a complete repertoire of human TCRB variable region segments operably linked to TCRBDJ1 and TCRBDJ2 clusters in which endogenous TCRA and/or TCRB non-coding sequences, e.g., non-coding DNA (e.g., non-coding recombination signal sequences (RSSs) and other non-coding intergenic sequences) that separates gene segments, may be retained.

The disclosure also provides a method of modifying a TCR variable gene locus (e.g., TCRα, TCRβ, TCRδ, and/or TCRγ gene locus) of a non-human animal to express a humanized TCR protein described herein. In one embodiment, the invention provides a method of modifying a TCR variable gene locus to express a humanized TCR protein on a surface of a T cell wherein the method comprises inserting, e.g., replacing, in a non-human animal an endogenous non-human TCR variable gene locus with an unrearranged humanized TCR variable gene locus. In one embodiment wherein the TCR variable gene locus is a TCRα variable gene locus, the unrearranged humanized TCR variable gene locus comprises at least one human Vα segment and at least one human Jα segment. In one embodiment wherein the TCR variable gene locus is a TCRβ variable gene locus, the unrearranged humanized TCR variable gene locus comprises at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment. In various aspects, the unrearranged humanized TCR variable gene locus is operably linked to the corresponding endogenous non-human TCR constant region.

Thus, nucleotide constructs for generating genetically modified animals comprising humanized TCR variable region genes are also provided. In one aspect, the nucleotide construct comprises: 5′ and 3′ homology arms, a human DNA fragment comprising human TCR variable region gene segment(s), and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a TCRα gene fragment and it comprises at least one human TCRα variable region segment. In another embodiment, the human DNA fragment is a TCRβ fragment and it comprises at least one human TCRβ variable region gene segment. In one aspect, at least one homology arm is a non-human homology arm and it is homologous to non-human TCR locus (e.g., non-human TCRα or TCRβ locus). In another embodiment, the human DNA fragment is a TCRβ fragment and it comprises at least one human TCRβ variable region gene segment. In one embodiment, the human DNA fragment is a TCRα gene fragment and it comprises at least one human TCRα variable region segment.

In some embodiments, the nucleotide construct comprises:

(i) 5′ and 3′ homology arms,

(ii) a nucleic acid sequence comprising a human TRBD1 and combination of human TRBJ1-1 to TRBJ1-6 gene segments and non-human (e.g., mouse) tcrbdj1 non-coding sequences (including non-coding recombination signal sequences (RSSs) and other non-intergenic sequences), where the human TRBD1 and human TRBJ1-1 to TRBJ1-6 gene segments flank the same non-human (e.g., mouse) tcrbdj1 TCR non-coding sequences as are normally flanked by non-human (e.g., mouse) Trbd1 and non-human (e.g., mouse) Trbj1-1 to Trbj1-6 gene segments, and/or

(iii) a nucleic acid sequence comprising a human TRBD2 and human TRBJ2-1 to TRBJ2-7 gene segments and mouse tcrbdj2 non-coding sequences, where the human TRBD2 and human TRBJ2-1 to TRBJ2-7 gene segments flank the same mouse Tcrbdj2 non-coding sequences as are normally flanked by the mouse Trbd2 and mouse Trbj2-1 to Trbj2-7 gene segments, and optionally,

(iv) a non-human (e.g., mouse) trbc1 gene sequence between the (ii) a nucleic acid sequence comprising a human TRBD1 and combination of human TRBJ1-1 to TRBJ1-6 gene segments and non-human (e.g., mouse) tcrbdj1 non-coding sequences (including non-coding recombination signal sequences (RSSs) and other non-intergenic sequences), and the (iii) a nucleic acid sequence comprising a human TRBD2 and human TRBJ2-1 to TRBJ2-7 gene segments and mouse tcrbdj2 non-coding sequences (including non-coding recombination signal sequences (RSSs) and other non-intergenic sequences) and/or

(v) selection cassette flanked by recombination sites.

In one aspect, at least one homology arm is a non-human homology arm and it is homologous to non-human TCR locus (e.g., non-human TCRα or TCRβ locus).

In various aspects of the invention, the sequence(s) encoding a chimeric human/non-human MHC I and MHC II polypeptides are located at an endogenous non-human MHC locus (e.g., mouse H-2K and/or H-2E locus). In one embodiment, this results in placement, e.g., replacement, of an endogenous MHC gene(s) or a portion thereof with a nucleic acid sequence(s) encoding a human or humanized MHC I polypeptides. Since the nucleic acid sequences encoding MHC I, MHC II α and MHC II β polypeptides are located in proximity to one another on the chromosome, in order to achieve the greatest success in humanization of both MHC I and MHC II in one animal, the MHC I and MHC II loci should be targeted sequentially. Thus, also provided herein are methods of generating a genetically modified non-human animal comprising nucleic acid sequences encoding chimeric human/non-human MHC I, MHC II α and MHC II β polypeptides as described herein.

Thus, a nucleotide construct for generating genetically modified animals comprising chimeric human/non-human MHC is provided. In one aspect, the nucleic acid construct comprises: 5′ and 3′ non-human homology arms, a human DNA fragment comprising human MHC gene sequences (e.g., human HLA-A2 or human HLA-DRs gene sequences), and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a genomic fragment that comprises both introns and exons of a human MHC gene (e.g., human HLA-A2 or HLA-DR2 gene). In one embodiment, the non-human homology arms are homologous to a non-human MHC locus (e.g., MHC I or MHC II locus).

In one embodiment, the 5′ and 3′ non-human homology arms comprise genomic sequence at 5′ and 3′ locations, respectively, of an endogenous non-human (e.g., murine) MHC class I or class II gene locus (e.g., 5′ of the first leader sequence and 3′ of the α3 exon of the mouse MHC I gene, or upstream of mouse H-2Ab1 gene and downstream of mouse H-2Ea gene). In one embodiment, the endogenous MHC class I locus is selected from mouse H-2K, H-2D and H-2L. In a specific embodiment, the endogenous MHC class I locus is mouse H-2K. In one embodiment, the endogenous MHC II locus is selected from mouse H-2E and H-2A. In one embodiment, the engineered MHC II construct allows replacement of both mouse H-2E and H-2A genes. In one embodiment, the mouse does not express functional endogenous MHC polypeptides from its H-2D locus. In some embodiments, the mouse is engineered to lack all or a portion of an endogenous H-2D locus. In another embodiment, the mouse does not express any functional endogenous MHC I and MHC II polypeptides on a cell surface. In one embodiment, the only MHC I and MHC II expressed by the mouse on a cell surface are chimeric human/mouse MHC I and MHC II.

The disclosure also provides methods for making a genetically engineered non-human animal (e.g., a genetically engineered rodent, e.g., a mouse or a rat) whose genome comprises a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide. In one aspect, the methods result in a genetically engineered rodent, e.g., mouse, whose genome comprises at an endogenous β2 microglobulin locus a nucleotide sequence encoding a human or humanized β2 microglobulin polypeptide. In some instances, the mouse does not express a functional mouse β2 microglobulin from an endogenous mouse β2 microglobulin locus. In some aspects, the methods utilize a targeting construct, e.g., made using VELOCIGENE® technology, introducing the construct into ES cells, and introducing targeted ES cell clones into a mouse embryo, e.g., using VELOCIMOUSE® technology, as described in herein.

Also provided is a nucleotide construct used for generating genetically engineered non-human animals. The nucleotide construct may comprise: 5′ and 3′ non-human homology arms, a human DNA fragment comprising human β2 microglobulin sequences, and a selection cassette flanked by recombination sites. In one embodiment, the human DNA fragment is a genomic fragment that comprises both introns and exons of a human β2 microglobulin gene. In one embodiment, the non-human homology arms are homologous to a non-human β2 microglobulin locus. The genomic fragment may comprise exons 2, 3, and 4 of the human β2 microglobulin gene. In one instance, the genomic fragment comprises, from 5′ to 3′: exon 2, intron, exon 3, intron, and exon 4, all of human β2 microglobulin sequence. The selection cassette may be located anywhere in the construct outside the β2 microglobulin coding region, e.g., it may be located 3′ of exon 4 of the human β2 microglobulin. The 5′ and 3′ non-human homology arms may comprise genomic sequence 5′ and 3′ of endogenous non-human β2 microglobulin gene, respectively. In another embodiment, the 5′ and 3′ non-human homology arms comprise genomic sequence 5′ of exon 2 and 3′ of exon 4 of endogenous non-human gene, respectively.

Another aspect of the invention relates to a method of modifying a β2 microglobulin locus of a non-human animal (e.g., a rodent, e.g., a mouse or a rat) to express a human or humanized β2 microglobulin polypeptide described herein. One method of modifying a β2 microglobulin locus of a non-human animal, e.g., mouse, to express a human or humanized β2 microglobulin polypeptide, comprises replacing at an endogenous β2 microglobulin locus a nucleotide sequence encoding a mouse β2 microglobulin with a nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide. In one embodiment of such method, the non-human animal, e.g., mouse does not express a functional β2 microglobulin polypeptide from an endogenous non-human, e.g., mouse β2 microglobulin locus. In some specific embodiments, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises nucleotide sequence set forth in exons 2 to 4 of the human β2 microglobulin gene. In other embodiments, the nucleotide sequence encoding the human or humanized β2 microglobulin polypeptide comprises nucleotide sequences set forth in exons 2, 3, and 4 of the human β2 microglobulin gene.

Various exemplary embodiments of the humanized loci described herein are presented in the figures and described in the examples.

Upon completion of gene targeting, ES cells or genetically modified non-human animals are screened to confirm successful incorporation of exogenous nucleotide sequence of interest or expression of exogenous polypeptide. Numerous techniques are known to those skilled in the art, and include (but are not limited to) Southern blotting, long PCR, quantitative PCR (e.g., real-time PCR using TAQMAN®), fluorescence in situ hybridization, Northern blotting, flow cytometry, Western analysis, immunocytochemistry, immunohistochemistry, etc. In one example, non-human animals (e.g., mice) bearing the genetic modification of interest can be identified by screening for loss of mouse allele and/or gain of human allele using a modification of allele assay described in Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-659. Other assays that identify a specific nucleotide or amino acid sequence in the genetically modified animals are known to those skilled in the art.

In some embodiments, animals are generated herein by breeding.

In one non-limiting aspect, for example, a non-human animal comprising the chimeric human/non-human CD8 described herein and the humanized MHC I and/or β2 microglobulin may be generated by breeding an animal comprising a chimeric CD8 locus (e.g., chimeric CD8 α and/or λ locus) as described herein with an animal comprising a humanized MHC I and/or β2 microglobulin locus. The animal may also be generated by introducing into ES cells of an animal comprising humanized MHC I and/or β2 microglobulin locus a nucleotide sequence encoding chimeric CD8 (e.g., chimeric CD8 α and/or β), e.g., for replacement at the endogenous CD8 locus (e.g., chimeric CD8 α and/or β locus); or introducing into ES cells of an animal comprising a chimeric CD8 locus (e.g., chimeric CD8 α and/or β locus) a nucleotide sequence(s) encoding humanized MHC I and/or β2 microglobulin.

In some embodiments, the animal comprising a chimeric CD8 locus may first be bred with an animal comprising a humanized TCR variable gene locus to create an animal comprising humanized CD8 and TCR variable region loci, which may then be bred with an animal comprising humanized MHC I and/or β2 microglobulin loci to generate an animal comprising humanized MHC I, TCR variable gene and/or β2 microglobulin loci. Alternatively, the animal comprising a humanized MHC I and/or β2 microglobulin loci may first be bred with an animal comprising a humanized TCR variable gene locus to create an animal comprising humanized MHC I and TCR variable region loci, which may then be bred with an animal comprising a chimeric CD8 locus generate an animal comprising humanized MHC I, TCR variable gene and/or β2 microglobulin loci, respectively.

In one aspect, the non-human animal comprising a chimeric human/non-human CD4 and the humanized MHC II may be generated by breeding an animal comprising a chimeric CD4 locus as described herein with an animal comprising a humanized MHC II locus. The animal may also be generated by introducing into ES cells of an animal comprising humanized MHC II locus a nucleotide sequence encoding chimeric CD4, e.g., for replacement at the endogenous CD4 locus; or introducing into ES cells of an animal comprising a chimeric CD4 locus a nucleotide sequence encoding humanized MHC II.

In some embodiments, the animal comprising a chimeric CD4 locus may first be bred with an animal comprising a humanized TCR variable gene locus to create an animal comprising humanized CD4 and TCR variable region loci, which may then be bred with an animal comprising a humanized MHC II locus to generate an animal comprising humanized CD4, MHC II and TCR variable gene loci. Alternatively, the animal comprising a comprising humanized MHC II locus may first be bred with an animal comprising a humanized TCR variable gene locus to create an animal comprising humanized MHC II and TCR variable region loci, which may then be bred with an animal comprising a chimeric CD4 locus generate an animal comprising humanized MHC II, TCR variable gene and/or β2 microglobulin loci, respectively.

In some embodiments, a non-human animal comprising the chimeric human/non-human CD8 described herein and the humanized MHC I and/or β2 microglobulin is bred with an animal comprising a chimeric CD4 locus as described herein and an animal comprising a humanized MHC II locus to generate a non-human animal comprising chimeric CD4 and CD8 polypeptides and humanized MHC I (and/or β2 microglobulin) and MHC II molecules. In some embodiments, the animal comprising chimeric human/non-human CD4 and CD8 polypeptides and humanized MHC I and MHC II molecules is bred with an animal comprising a humanized TCR variable domain to generate an animal comprising a substantially humanized T cell immune system, e.g., chimeric human/non-human CD4 and CD8 polypeptides, humanized MHC I (and/or β2 microglobulin) and MHC II molecules and humanized TCR variable domains.

Any of the genetically modified no-human animal (e.g., mouse) described herein may comprise one or two copies of the genes encoding chimeric human/non-human CD8 (e.g., CD8α and/or CD8β); chimeric human/non-human CD4; human or humanized MHC I; human or humanized β2 microglobulin; human or humanized MHC II (e.g., MHC IIα and/or MHC IIβ); and human or humanized TCR (e.g., TCR α and/or TCRβ). Accordingly, the animal may be heterozygous or homozygous for any or all of these genes.

Using Genetically Modified Non-Human Animals that Mount Substantially Humanized T Cell Immune Responses

The genetically modified non-human animals, e.g., rodents, e.g., mice or rats, comprising either humanized CD4 and MHC II or humanized CD8 and MHC I (and β2 microglobulin), or both, present peptides to T cells (CD4+ or CD8+ T cells, respectively) in a human-like manner, because substantially all of the components of the complex are human or humanized. The genetically modified non-human animals of the invention can be used to study the function of a human immune system in the humanized animal; for identification of antigens and antigen epitopes that elicit immune response (e.g., T cell epitopes, e.g., unique human cancer epitopes), e.g., for use in vaccine development; for identification of high affinity T cells to human pathogens or cancer antigens (i.e., T cells that bind to antigen in the context of human MHC I complex with high avidity), e.g., for use in adaptive T cell therapy; for evaluation of vaccine candidates and other vaccine strategies; for studying human autoimmunity; for studying human infectious diseases; and otherwise for devising better therapeutic strategies based on human MHC and CD4/CD8 expression.

Thus, in various embodiments, the genetically engineered animals of the present invention are useful, among other things, for evaluating the capacity of an antigen to initiate an immune response in a human, and for generating a diversity of antigens and identifying a specific antigen that may be used in human vaccine development.

In one aspect, a method for determining whether a peptide will provoke a cellular immune response in a human is provided, comprising exposing a genetically modified non-human animal as described herein to the peptide, allowing the non-human animal to mount an immune response, and detecting in the non-human animal a cell (e.g., a CD8+ or CD4+ T cell, comprising a human CD8 or CD4, respectively) that binds a sequence of the peptide presented by a chimeric human/non-human MHC I or II molecule as described herein. In one embodiment, the non-human animal following exposure comprises an MHC class I-restricted CD8+ cytotoxic T lymphocyte (CTL) that binds the peptide. In another embodiment, the non-human animal following exposure comprises an MHC II-restricted CD4+ T cell that binds the peptide.

In one aspect, a method for identifying a human T cell epitope is provided, comprising exposing a non-human animal as described herein to an antigen comprising a putative T cell epitope, allowing the non-human animal to mount an immune response, isolating from the non-human animal an MHC class I- or MHC class II-restricted T cell that binds the epitope, and identifying the epitope bound by said T cell.

In one aspect, a method is provided for identifying an antigen that generates a T cell response in a human, comprising exposing a putative antigen to a mouse as described herein, allowing the mouse to generate an immune response, and identifying the antigen bound by the HLA class I- or class II-restricted molecule.

In one aspect, a method is provided for determining whether a putative antigen contains an epitope that upon exposure to a human immune system will generate an HLA class I- or class II-restricted immune response, comprising exposing a mouse as described herein to the putative antigen and measuring an antigen-specific HLA class I- or HLA class II-restricted immune response in the mouse.

In addition, the genetically engineered non-human animals described herein may be useful for identification of T cell receptors, e.g., high-avidity T cell receptors, that recognize an antigen of interest, e.g., a tumor or another disease antigen. The method may comprise: exposing the non-human animal described herein to an antigen, allowing the non-human animal to mount an immune response to the antigen, isolating from the non-human animal a T cell comprising a T cell receptor that binds the antigen presented by a human or humanized MHC I or MHC II, and determining the sequence of said T cell receptor.

Non-human animals expressing a diverse repertoire of functional human TCR V(D)J gene segments may be useful for the study of human diseases. Accordingly, in one embodiment, the genetically engineered non-human animals described herein may express a TCR repertoire substantially similar to a TCR repertoire expressed in a human, e.g., the TCR repertoire of a non-human animal disclosed herein may be derived from at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of all functional human TCR α, TCR R, TCRγ and/or TCRδ gene segments. In some embodiments, a non-human animal as disclosed expresses a TCR repertoire derived from

(i) at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of all functional human TCR Vα gene segments;

(ii) at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of all functional human TCR Jα gene segments;

(iii) at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of all functional human TCR Vβ gene segments;

(iv) at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of all functional human TCR Dβ gene segments; and/or

(v) at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of all functional human TCR Jβ gene segments.

In one embodiment, the mouse produces a T cell repertoire comprising all or substantially all functional human TCR Vα gene segments, and comprising all or substantially all functional human TCR Vβ gene segments. In one embodiment, the mouse provided herein utilizes human TCR Vα and/or Vβ genes with a frequency similar to the frequency of human TCR Vα and/or Vβ genes, respectively, utilized by human T cells in a human. Methods of detecting the gene segments expressed in the TCR repertoire of the non-human animal include, e.g., flow cytometric and/or sequencing methods (e.g., real time PCR, Next Generation Sequencing, etc.).

In one embodiment, a method is provided for determining T cell activation by a putative human therapeutic, comprising exposing a genetically modified animal as described herein to a putative human therapeutic (or e.g., exposing a human or humanized MHC II- or MHC I-expressing cell of such an animal to a peptide sequence of the putative therapeutic), exposing a cell of the genetically modified animal that displays a human or humanized MHC/peptide complex to a T cell comprising a chimeric human/non-human (e.g., human/mouse) CD4 or CD8 capable of binding the cell of the genetically modified animal, and measuring activation of the T cell that is induced by the peptide-displaying cell of the genetically modified animal.

In addition to the ability to identify antigens and antigen epitopes from human pathogens or neoplasms, the genetically modified animals of the invention can be used to identify autoantigens of relevance to human autoimmune diseases, e.g., type I diabetes, multiple sclerosis, etc. Also, the genetically modified animals of the invention can be used to study various aspects of human autoimmune disease, and may be utilized as autoimmune disease models.

In various embodiments, the genetically modified non-human animals of the invention make T cells with humanized TCR molecules on their surface, and as a result, would recognize peptides presented to them by MHC complexes in a human-like manner. The genetically modified non-human animals described herein may be used to study the development and function of human T cells and the processes of immunological tolerance; to test human vaccine candidates; to generate TCRs with certain specificities for TCR gene therapy; to generate TCR libraries to disease associated antigens (e.g., tumor associated antigens (TAAs); etc.

There is a growing interest in T cell therapy in the art, as T cells (e.g., cytotoxic T cells) can be directed to attack and lead to destruction of antigen of interest, e.g., viral antigen, bacterial antigen, tumor antigen, etc., or cells that present it. Initial studies in cancer T cell therapy aimed at isolation of tumor infiltrating lymphocytes (TILs; lymphocyte populations in the tumor mass that presumably comprise T cells reactive against tumor antigens) from tumor cell mass, expanding them in vitro using T cell growth factors, and transferring them back to the patient in a process called adoptive T cell transfer. See, e.g., Restifo et al. (2012) Adoptive immunotherapy for cancer: harnessing the T cell response, Nature Reviews 12:269-81; Linnermann et al. (2011) T-Cell Receptor Gene Therapy: Critical Parameters for Clinical Success, J. Invest. Dermatol. 131:1806-16. However, success of these therapies has thus far been limited to melanoma and renal cell carcinoma; and the TIL adoptive transfer is not specifically directed to defined tumor associated antigens (TAAs). Linnermann et al., supra.

Attempts have been made to initiate TCR gene therapy where T cells are either selected or programmed to target an antigen of interest, e.g., a TAA. Current TCR gene therapy relies on identification of sequences of TCRs that are directed to specific antigens, e.g., tumor associated antigens. For example, Rosenberg and colleagues have published several studies in which they transduced peripheral blood lymphocytes derived from a melanoma patient with genes encoding TCRα and β chains specific for melanoma-associated antigen MART-1 epitopes, and used resulting expanded lymphocytes for adoptive T cell therapy. Johnson et al. (2009) Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen, Blood 114:535-46; Morgan et al. (2006) Cancer Regression in Patients After Transfer of Genetically Engineered Lymphocytes, Science 314:126-29. The MART-1 specific TCRs were isolated from patients that experienced tumor regression following TIL therapy. However, identification of such TCRs, particularly high-avidity TCRs (which are most likely to be therapeutically useful), is complicated by the fact that most tumor antigens are self-antigens, and TCRs targeting these antigens are often either deleted or possess suboptimal affinity, due primarily to immunological tolerance.

In various embodiments, the present invention solves this problem by providing genetically engineered non-human animals comprising in their genome an unrearranged human TCR variable gene locus. The non-human animal described herein is capable of generating T cells with a diverse repertoire of humanized T cell receptors. Thus, the non-human animals described herein may be a source of a diverse repertoire of humanized T cell receptors, e.g., high-avidity humanized T cell receptors for use in adoptive T cell transfer.

Thus, in one embodiment, the present invention provides a method of generating a T cell receptor to a human antigen comprising immunizing a non-human animal (e.g., a rodent, e.g., a mouse or a rat) described herein with an antigen of interest, allowing the animal to mount an immune response, isolating from the animal an activated T cell with specificity for the antigen of interest, and determining the nucleic acid sequence of the T cell receptor expressed by the antigen-specific T cell.

In one embodiment, the invention provides a method of producing a human T cell receptor specific for an antigen of interest (e.g., a disease-associated antigen) comprising immunizing a non-human animal described herein with the antigen of interest; allowing the animal to mount an immune response; isolating from the animal a T cell reactive to the antigen of interest; determining a nucleic acid sequence of a human TCR variable region expressed by the T cell; cloning the human TCR variable region into a nucleotide construct comprising a nucleic acid sequence of a human TCR constant region such that the human TCR variable region is operably linked to the human TCR constant region; and expressing from the construct a human T cell receptor specific for the antigen of interest. In one embodiment, the steps of isolating a T cell, determining a nucleic acid sequence of a human TCR variable region expressed by the T cell, cloning the human TCR variable region into a nucleotide construct comprising a nucleic acid sequence of a human TCR constant region, and expressing a human T cell receptor are performed using standard techniques known to those of skill the art.

In one embodiment, the nucleotide sequence encoding a T cell receptor specific for an antigen of interest is expressed in a cell. In one embodiment, the cell expressing the TCR is selected from a CHO, COS, 293, HeLa, PERC.6™ cell, etc.

The antigen of interest may be any antigen that is known to cause or be associated with a disease or condition, e.g., a tumor associated antigen; an antigen of viral, bacterial or other pathogenic origin; etc. Many tumor-associated antigens are known in the art. A selection of tumor associated antigens is presented in Cancer Immunity (A Journal of the Cancer Research Institute) Peptide Database (archive.cancerimmunity.org/peptidedatabase/Tcellepitopes.htm). In some embodiments of the invention, the antigen of interest is a human antigen, e.g., a human tumor associated antigen. In some embodiments, the antigen is a cell type-specific intracellular antigen, and a T cell receptor is used to kill a cell expressing the antigen.

In one embodiment, provided herein is a method of identifying a T cell with specificity against an antigen of interest, e.g., a tumor associated antigen, comprising immunizing a non-human animal described herein with the antigen of interest, allowing the animal to mount an immune response, and isolating from the non-human animal a T cell with specificity for the antigen.

The present invention provides new methods for adoptive T cell therapy. Thus, provided herein is a method of treating or ameliorating a disease or condition (e.g., a cancer) in a subject (e.g., a mammalian subject, e.g., a human subject) comprising immunizing a non-human animal described herein with an antigen associated with the disease or condition, allowing the animal to mount an immune response, isolating from the animal a population of antigen-specific T cells, and infusing isolated antigen-specific T cells into the subject. In one embodiment, the invention provides a method of treating or ameliorating a disease or condition in a human subject, comprising immunizing the non-human animal described herein with an antigen of interest (e.g., a disease- or condition-associated antigen, e.g., a tumor associated antigen), allowing the animal to mount an immune response, isolating from the animal a population of antigen-specific T cells, determining the nucleic acid sequence of a T cell receptor, (e.g., a first and/or second nucleic acid sequence encoding the human rearranged TCRα and/or human rearranged TCRβ variable region gene); a third and/or fourth nucleic acid sequence encoding the human rearranged TCRδ variable region gene or a TCRγ variable region gene, expressed by the antigen-specific T cells, cloning the nucleic acid sequence of the T cell receptor, e.g., the first, second, third and/or fourth nucleic acid sequence respectively encoding the human rearranged TCRα variable region gene, human rearranged TCRβ variable region gene, TCRδ variable region gene or a TCRγ variable region gene, into an expression vector (e.g., a retroviral vector), e.g., optionally wherein the first, second, third and/or fourth nucleic acid sequence respectively encoding the human rearranged TCRα variable region gene, human rearranged TCRβ variable region gene, TCRδ variable region gene or a TCRγ variable region gene is respectively cloned in-frame with a human TCRα constant gene, human TCRβ constant gene, TCRδ constant gene or a TCRγ constant gene, introducing the vector into T cells derived from the subject such that the T cells express the antigen-specific T cell receptor, and infusing the T cells into the subject. In one embodiment, the T cell receptor nucleic acid sequence is further humanized prior to introduction into T cells derived from the subject, e.g., the sequence encoding the non-human constant region is modified to further resemble a human TCR constant region (e.g., the non-human constant region is replaced with a human constant region). In some embodiments, the disease or condition is cancer. In some embodiments, an antigen-specific T cell population is expanded prior to infusing into the subject. In some embodiments, the subject's immune cell population is immunodepleted prior to the infusion of antigen-specific T cells. In some embodiments, the antigen-specific TCR is a high avidity TCR, e.g., a high avidity TCR to a tumor associated antigen. In some embodiments, the T cell is a cytotoxic T cell. In other embodiments, the disease or condition is caused by a virus or a bacterium.

In another embodiment, a disease or condition is an autoimmune disease. TREG cells are a subpopulation of T cells that maintain tolerance to self-antigens and prevent pathological self-reactivity. Thus, also provided herein are methods of treating autoimmune disease that rely on generation of antigen-specific TREG cells in the non-human animal of the invention described herein.

Also provided herein is a method of treating or ameliorating a disease or condition (e.g., a cancer) in a subject comprising introducing the cells affected by the disease or condition (e.g., cancer cells) from the subject into a non-human animal, allowing the animal to mount an immune response to the cells, isolating from the animal a population of T cells reactive to the cells, determining the nucleic acid sequence of a T cell receptor variable domain expressed by the T cells, cloning the T cell receptor variable domain encoding sequence into a vector (e.g., in-frame and operably linked to a human TCR constant gene), introducing the vector into T cells derived from the subject, and infusing the subject's T cells harboring the T cell receptor into the subject.

Also provided herein is the use of a non-human animal as described herein to make nucleic acid sequences encoding human TCR variable domains (e.g., TCR α and/or β variable domains). In one embodiment, a method is provided for making a nucleic acid sequence encoding a human TCR variable domain, comprising immunizing a non-human animal as described herein with an antigen of interest, allowing the non-human animal to mount an immune response to the antigen of interest, and obtaining therefrom a nucleic acid sequence encoding a human TCR variable domain that binds the antigen of interest. In one embodiment, the method further comprises making a nucleic acid sequence encoding a human TCR variable domain, that is optionally operably linked to a non-human TCR constant region, comprising isolating a T cell from a non-human animal described herein and obtaining therefrom the nucleic acid sequence encoding the TCR variable domain, optionally linked to the non-human constant region TCR constant region, and cloning the nucleic acid sequence(s) encoding the TCR variable domain (e.g., a first, second, third or fourth nucleic acid sequence respectively encoding a human rearranged TCRα variable region gene, human rearranged TCRβ variable region gene, TCRδ variable region gene or a TCRγ variable region gene) in-frame with an appropriate human constant region (e.g., a human TCRα constant region gene, human TCRβ constant region gene, TCRδ constant region gene or a TCRγ variable region gene, respectively).

Thus, provided herein are TCR variable region nucleic acid sequences, such as rearranged TCR variable nucleic acid sequences, e.g., rearranged TCRα and/or TCRβ variable region nucleic acid sequences, generated in the non-human animals described herein, and encoded respectively by, e.g., a human rearranged Vα/Jα gene sequence and a rearranged human VβDβJβ gene sequence. Also, provided are TCR variable region amino acid sequences encoded by such rearranged TCR variable region nucleic acid sequences. Such rearranged TCR variable region nucleic acid sequences (TCRα and/or TCRβ variable region nucleic acid sequences) obtained in the non-human animals described herein may be cloned in operable linkage with human TCR constant region (TCRα and/or TCRβ constant region), and utilized for various uses described herein, e.g., as a human therapeutic, in a human.

Also provided herein is the use of a non-human animal as described herein to make a human therapeutic, comprising immunizing the non-human animal with an antigen of interest (e.g., a tumor associated antigen), allowing the non-human animal to mount an immune response, obtaining from the animal T cells reactive to the antigen of interest, obtaining from the T cells a nucleic acid sequence(s) encoding a humanized TCR protein or human TCR variable domain that binds the antigen of interest, and employing the nucleic acid sequence(s) encoding a humanized TCR protein or a human TCR variable domain in a human therapeutic.

Thus, also provided is a method for making a human therapeutic, comprising immunizing a non-human animal as described herein with an antigen of interest, allowing the non-human animal to mount an immune response, obtaining from the animal T cells reactive to the antigen of interest, obtaining from the T cells a nucleic acid sequence(s) encoding a humanized T cell receptor that binds the antigen of interest, and employing the humanized (or fully human) T cell receptor in a human therapeutic.

In one embodiment, the human therapeutic is a T cell (e.g., a human T cell, e.g., a T cell derived from a human subject) harboring a nucleic acid sequence of interest (e.g., transfected or transduced or otherwise introduced with the nucleic acid of interest) such that the T cell expresses the humanized TCR protein with affinity for an antigen of interest. In one aspect, a subject in whom the therapeutic is employed is in need of therapy for a particular disease or condition, and the antigen is associated with the disease or condition. In one aspect, the T cell is a cytotoxic T cell, the antigen is a tumor associated antigen, and the disease or condition is cancer. In one aspect, the T cell is derived from the subject.

In another embodiment, the human therapeutic is a T cell receptor. In one embodiment, the therapeutic receptor is a soluble T cell receptor. Much effort has been expanded to generate soluble T cell receptors or TCR variable regions for use therapeutic agents. Generation of soluble T cell receptors depends on obtaining rearranged TCR variable regions. One approach is to design single chain TCRs comprising TCRα and TCRβ, and, similarly to scFv immunoglobulin format, fuse them together via a linker (see, e.g., International Application No. WO 2011/044186). The resulting scTv, if analogous to scFv, would provide a thermally stable and soluble form of TCRα/β binding protein. Alternative approaches included designing a soluble TCR having TCRβ constant domains (see, e.g., Chung et al., (1994) Functional three-domain single-chain T-cell receptors, Proc. Natl. Acad. Sci. USA. 91:12654-58); as well as engineering a non-native disulfide bond into the interface between TCR constant domains (reviewed in Boulter and Jakobsen (2005) Stable, soluble, high-affinity, engineered T cell receptors: novel antibody-like proteins for specific targeting of peptide antigens, Clinical and Experimental Immunology 142:454-60; see also, U.S. Pat. No. 7,569,664). Other formats of soluble T cell receptors have been described. The non-human animals described herein may be used to determine a sequence of a T cell receptor that binds with high affinity to an antigen of interest, and subsequently design a soluble T cell receptor based on the sequence.

A soluble T cell receptor derived from the TCR receptor sequence expressed by the non-human animal can be used to block the function of a protein of interest, e.g., a viral, bacterial, or tumor associated protein. Alternatively, a soluble T cell receptor may be fused to a moiety that can kill an infected or cancer cell, e.g., a cytotoxic molecule (e.g., a chemotherapeutic), toxin, radionuclide, prodrug, antibody, etc. A soluble T cell receptor may also be fused to an immunomodulatory molecule, e.g., a cytokine, chemokine, etc. A soluble T cell receptor may also be fused to an immune inhibitory molecule, e.g., a molecule that inhibits a T cell from killing other cells harboring an antigen recognized by the T cell. Such soluble T cell receptors fused to immune inhibitory molecules can be used, e.g., in blocking autoimmunity. Various exemplary immune inhibitory molecules that may be fused to a soluble T cell receptor are reviewed in Ravetch and Lanier (2000) Immune Inhibitory Receptors, Science 290:84-89, incorporated herein by reference.

The present invention also provides methods for studying immunological response in the context of human TCR, including human TCR rearrangement, T cell development, T cell activation, immunological tolerance, etc.

Also provided are methods of testing vaccine candidates. In one embodiment, provided herein is a method of determining whether a vaccine will activate an immunological response (e.g., T cell proliferation, cytokine release, etc.), and lead to generation of effector, as well as memory T cells (e.g., central and effector memory T cells).

In one aspect, an in vitro preparation is provided that comprises a T cell that bears a chimeric CD8 protein on its surface and a second cell that binds the chimeric CD8. In one embodiment, the second cell is a cell expressing an MHC I polypeptide, e.g., a chimeric human/non-human MHC I protein. In one embodiment, the chimeric CD8 on the surface of the first cell interacts with chimeric MHC I on the surface of the second cell. In one embodiment, the chimeric CD8 protein retains interaction with endogenous cytosolic molecules, e.g., endogenous cytosolic signaling molecules (e.g., endogenous Lck, etc.).

In one aspect, an in vitro preparation is provided that comprises a T cell that bears a chimeric CD4 protein on its surface and a second cell that binds the chimeric CD4. In one embodiment, the second cell is a cell, e.g., an APC, expressing an MHC II polypeptide, e.g., a chimeric human/non-human MHC II protein. In one embodiment, the chimeric CD4 on the surface of the first cell interacts with chimeric MHC II on the surface of the second cell. In one embodiment, the chimeric CD4 protein retains interaction with endogenous cytosolic molecules, e.g., endogenous cytosolic signaling molecules (e.g., endogenous Lck, etc.).

Non-limiting and exemplary embodiments are provided below.

-   -   Embodiment 1. A mouse or an isolated mouse cell comprising:         -   (A) an unrearranged T cell receptor (TCR) α variable region             sequence comprising at least one unrearranged human T cell             variable region Vα segment and at least one unrearranged             human T cell variable region Jα segment operably linked to a             mouse TCR α constant gene sequence, optionally at an             endogenous mouse TCR α variable gene locus, wherein the             unrearranged TCR α variable region sequence comprises a             mouse TCRA non-coding sequence, or         -   (B) an unrearranged TCRβ variable region sequence comprising             at least one unrearranged human T cell variable region Vβ             segment, at least one unrearranged human T cell variable             region Dβ segment, and at least one unrearranged human T             cell variable region Jβ segment operably linked to a mouse             TCRβ constant gene sequence, optionally at an endogenous             mouse TCRβ variable gene locus, wherein the unrearranged             TCRβ variable region sequence comprises a mouse TCRB             non-coding sequence, or         -   (C) (i) an unrearranged T cell receptor (TCR) α variable             region sequence comprising at least one unrearranged human T             cell variable region Vα segment and at least one             unrearranged human T cell variable region Jα segment             operably linked to a mouse TCR α constant gene sequence,             optionally at an endogenous mouse TCR α variable gene locus,             wherein the unrearranged TCR α variable region sequence             comprises a mouse TCRA non-coding sequence, and             -   (ii) an unrearranged TCRβ variable region sequence                 comprising at least one unrearranged human T cell                 variable region Vβ segment, at least one unrearranged                 human T cell variable region Dβ segment, and at least                 one unrearranged human T cell variable region Jβ segment                 operably linked to a mouse TCRβ constant gene sequence,                 optionally at an endogenous mouse TCRβ variable gene                 locus, wherein the unrearranged TCRβ variable region                 sequence comprises a mouse TCRB non-coding sequence,         -   wherein the unrearranged human T cell variable region             segments are capable of rearranging in a T cell to form             genes that encode human T cell receptor variable domains             that specifically bind an antigen of interest.     -   Embodiment 2. The mouse or isolated mouse cell of embodiment 1,         wherein:         -   (A) the at least one unrearranged human T cell variable             region Vα segment comprises a repertoire of unrearranged             human T cell variable region Vα segments and the at least             one unrearranged human T cell variable region Jα segment             comprises a repertoire of unrearranged human T cell variable             region Jα gene segments, or         -   (B) the at least one unrearranged human T cell variable             region Vβ segment comprises a repertoire of unrearranged             human Vβ segments, the at least one unrearranged human T             cell variable region Dβ segment comprises a repertoire of             unrearranged human Dβ segments, and the at least one             unrearranged human T cell variable region Jβ segment             comprises a repertoire of unrearranged human Jβ gene             segments, or         -   (C) (i) the at least one unrearranged human T cell variable             region Vα segment comprises a repertoire of unrearranged             human T cell variable region Vα segments, the at least one             unrearranged human T cell variable region Jα segment             comprises a repertoire of unrearranged human T cell variable             region Jα gene segments,             -   (ii) the at least one unrearranged human T cell variable                 region Vβ segment comprises a repertoire of unrearranged                 human Vβ segments, the at least one unrearranged human T                 cell variable region Dβ segment comprises an                 unrearranged human T cell variable region Dβ1 segment                 and an unrearranged human T cell variable region Dβ2                 segment, and the at least one unrearranged human T cell                 variable region Jβ segment comprises at least one                 unrearranged human T cell variable region Jβ1 segment                 and at least one unrearranged human T cell variable                 region Jβ2 segment,         -   wherein the mouse TCRB non-coding sequence comprises (i) a             mouse TCRBD1-TCRBJ1 non-coding nucleic acid sequence between             the at least one unrearranged human T cell variable region             Dβ1 segment and the at least one unrearranged human T cell             variable region Jβ1 segment and (ii) a mouse TCRBD2-TCRBJ2             non-coding nucleic acid sequence between the at least one             unrearranged human T cell variable region Dβ2 segment and             the at least one unrearranged human T cell variable region             Jβ2 segment.     -   Embodiment 3. The mouse or isolated mouse cell of embodiment 1         or embodiment 2, wherein:         -   (I) the endogenous mouse TCRα variable gene locus comprises             a deletion selected from the group consisting of             -   (a) a deletion of all endogenous TCR Vα gene segments,             -   (b) a deletion of all endogenous TCR Jα gene segments,                 and             -   (c) a combination thereof, or         -   (II) the endogenous mouse TCRβ variable gene locus comprises             a deletion selected from the group consisting of             -   (a) a deletion of all endogenous TCR Vβ gene segments,             -   (b) a deletion of all endogenous TCR Dβ gene segments,             -   (c) a deletion of all endogenous TCR Jβ gene segments,                 and             -   (d) a combination thereof, or         -   (III) the endogenous mouse TCRα variable gene locus             comprises a deletion selected from the group consisting of             -   (a) a deletion of all endogenous TCR Vα gene segments,             -   (b) a deletion of all endogenous TCR Jα gene segments,                 and             -   (c) a combination thereof, and         -   the endogenous mouse TCRβ variable gene locus comprises a             deletion selected from the group consisting of             -   (a) a deletion of all endogenous TCR Vβ gene segments,             -   (b) a deletion of all endogenous TCR Dβ gene segments,             -   (c) a deletion of all endogenous TCR Jβ gene segments,                 and             -   (d) a combination thereof.     -   Embodiment 4. The mouse or isolated mouse cell of any one of         embodiments 1-3, wherein         -   (I) the endogenous mouse TCRα variable gene locus comprises             a replacement selected from the group consisting of             -   (a) a replacement at least one endogenous T cell                 variable region Vα gene segment with the at least one                 unrearranged human T cell variable region Vα gene                 segment,             -   (b) a replacement of at least one endogenous T cell                 variable region Jα gene segments with the at least                 unrearranged human T cell variable region Jα segment,                 and             -   (c) a combination thereof, or         -   (II) the endogenous mouse TCRβ variable gene locus comprises             a replacement selected from the group consisting of             -   (a) a replacement of at least one endogenous T cell                 variable region Vβ gene segment with the at least one                 unrearranged human T cell variable region Vβ segment,             -   (b) a replacement of at least one endogenous T cell                 variable region Dβ gene segment with the at least one                 unrearranged human T cell variable region Dβ segment,             -   (c) a replacement of at least one endogenous T cell                 variable region Jβ gene segment with the at least one                 unrearranged human T cell variable region Jβ segment,                 and             -   (d) a combination thereof, or         -   (III) the endogenous mouse TCRα variable gene locus             comprises a replacement selected from the group consisting             of             -   (a) a replacement at least one endogenous T cell                 variable region Vα gene segment with the at least one                 unrearranged human T cell variable region Vα gene                 segment,             -   (b) a replacement of at least one endogenous T cell                 variable region Jα gene segments with the at least                 unrearranged human T cell variable region Jα segment,                 and             -   (c) a combination thereof, and         -   the endogenous mouse TCRβ variable gene locus comprises a             replacement selected from the group consisting of             -   (a) a replacement of at least one endogenous T cell                 variable region Vβ gene segment with the at least one                 unrearranged human T cell variable region Vβ segment,             -   (b) a replacement of at least one endogenous T cell                 variable region Dβ gene segment with the at least one                 unrearranged human T cell variable region Dβ segment,             -   (c) a replacement of at least one endogenous T cell                 variable region Jβ gene segment with the at least one                 unrearranged human T cell variable region Jβ segment,                 and             -   (d) a combination thereof.     -   Embodiment 5. The mouse or isolated mouse cell of any one of         embodiments 1-4, wherein         -   (I) the endogenous mouse TCRα variable gene locus comprises:             -   (a) a replacement of all endogenous T cell variable                 region Vα gene segment with the at least one                 unrearranged human T cell variable region Vα gene                 segment, optionally wherein the at least one                 unrearranged human T cell variable region Vα gene                 segment comprises a plurality or all unrearranged human                 T cell variable region Vα gene segments from TRAV1-1 to                 TRAV41,             -   (b) a replacement of all endogenous T cell variable                 region Jα gene segments with the at least one                 unrearranged human T cell variable region Jα segment,                 optionally wherein the at least one unrearranged human T                 cell variable region Jα segment comprises a plurality or                 all unrearranged human T cell variable region Jα gene                 segments from TRAJ1 to TRAJ61, or             -   (c) a combination thereof,         -   (II) the endogenous mouse TCRβ variable gene locus comprises             -   (a) a replacement of all contiguous endogenous T cell                 variable region Vβ gene segments with the at least one                 unrearranged human T cell variable region Vβ segment,                 optionally wherein the at least one unrearranged human T                 cell variable region Vβ gene segment comprises a                 plurality or all unrearranged human T cell variable                 region Vβ gene segments from TRBV1 to TRBV29-1,             -   (b) a replacement of all endogenous T cell variable                 region DR gene segments with the at least one                 unrearranged human T cell variable region Dβ gene                 segment, optionally wherein the at least one                 unrearranged human T cell variable region Dβ gene                 segment comprises an unrearranged human T cell variable                 region Dβ1 gene segment and/or an unrearranged human T                 cell variable region Dβ2 gene segment,             -   (c) a replacement of all endogenous T cell variable                 region Jβ gene segments with the at least one                 unrearranged human T cell variable region Jβ segment,                 optionally wherein the at least one unrearranged human T                 cell variable region Jβ segment comprises a plurality or                 all unrearranged human Jβ segment from TRBJ1-1 to                 TRBJ1-6 and/or a plurality or all unrearranged human Jβ                 segments from TRBJ2-1 to TRBJ2-7, or             -   (d) a combination thereof, or         -   (III) the endogenous mouse TCRα variable gene locus             comprises             -   (a) a replacement of all endogenous T cell variable                 region Vα gene segments with the at least one                 unrearranged human T cell variable region Vα gene                 segment, optionally wherein the at least one                 unrearranged human T cell variable region Vα gene                 segment comprises a plurality or all unrearranged human                 T cell variable region gene segments from TRAV1-1 to                 TRAV41,             -   (b) a replacement of all endogenous T cell variable                 region Jα gene segments with the at least one                 unrearranged human T cell variable region Jα segment,                 optionally wherein the at least one unrearranged human T                 cell variable region Jα segment comprises a plurality or                 all unrearranged human T cell variable region gene                 segments from TRAJ1 to TRAJ61, or             -   (c) a combination thereof, and         -   the endogenous mouse TCRβ variable gene locus comprises             -   (a) a replacement of all contiguous endogenous T cell                 variable region Vβ gene segments with the at least one                 unrearranged human T cell variable region Vβ segment,                 optionally wherein the at least one unrearranged human T                 cell variable region Vβ gene segment comprises a                 plurality or all unrearranged human T cell variable                 region gene segments from TRBV1 to TRBV29-1,             -   (b) a replacement of all endogenous T cell variable                 region DR gene segments with the at least one                 unrearranged human T cell variable region Dβ gene                 segment, optionally wherein the at least one                 unrearranged human T cell variable region Dβ gene                 segment comprises an unrearranged human T cell variable                 region Dβ1 gene segment and/or an unrearranged human T                 cell variable region Dβ2 gene segment,             -   (c) a replacement of all endogenous T cell variable                 region Jβ gene segments with the at least one                 unrearranged human T cell variable region Jβ segment,                 optionally wherein the at least one unrearranged human T                 cell variable region Jβ segment comprises a plurality or                 all unrearranged human Jβ segments from TRBJ1-1 to                 TRBJ1-6 and/or a plurality or all unrearranged human Jβ                 segments from TRBJ2-1 to TRBJ2-7, or             -   (d) a combination thereof.     -   Embodiment 6. The mouse or isolated mouse cell of any one of         embodiments 1-5, wherein         -   (I) the endogenous mouse TCRα variable gene locus comprises:             -   (a) a replacement of all endogenous T cell variable                 region Vα gene segments with all unrearranged human T                 cell variable region Vα gene segments from TRAV1-1 to                 TRAV41,             -   (b) a replacement of all endogenous T cell variable                 region Jα gene segments with all unrearranged human T                 cell variable region Jα gene segments from TRAJ1 to                 TRAJ61, or             -   (c) a combination thereof,         -   (II) the endogenous mouse TCRβ variable gene locus comprises             -   (a) a replacement of all contiguous endogenous T cell                 variable region Vβ gene segments with all unrearranged                 human T cell variable region Vβ gene segments from TRBV1                 to TRBV29-1,             -   (b) a replacement of endogenous T cell variable region                 Dβ1 gene segment with unrearranged human T cell variable                 region Dβ1 gene segment and a replacement of endogenous                 T cell variable region Dβ2 gene segment with                 unrearranged human T cell variable region Dβ2 gene                 segment,             -   (c) a replacement of                 -   an endogenous TRBJ1-1 gene segment with an                     unrearranged human TRBJ1-1 gene segment,                 -   an endogenous TRBJ1-2 gene segment with an                     unrearranged human TRBJ1-2 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ1-1 segment and a mouse TRBJ1-2 segment,                 -   an endogenous TRBJ1-3 gene segment with an                     unrearranged human TRBJ1-3 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ1-2 segment and a mouse TRBJ1-3 segment,                 -   an endogenous TRBJ1-4 gene segment with an                     unrearranged human TRBJ1-4 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ1-3 segment and a mouse TRBJ1-4 segment,                 -   an endogenous TRBJ1-5 gene segment with an                     unrearranged human TRBJ1-5 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ1-4 segment and a mouse TRBJ1-5 segment,                 -   an endogenous TRBJ1-6 gene segment with an                     unrearranged human TRBJ1-6 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ1-5 segment and a mouse TRBJ1-6 segment,                 -   an endogenous TRBJ2-1 gene segment with an                     unrearranged human TRBJ2-1 gene segment,                 -   an endogenous TRBJ2-2 gene segment with an                     unrearranged human TRBJ2-2 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ2-1 segment and a mouse TRBJ2-2 segment,                 -   an endogenous TRBJ2-3 gene segment with an                     unrearranged human TRBJ2-3 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ2-2 segment and a mouse TRBJ2-3 segment,                 -   an endogenous TRBJ2-4 gene segment with an                     unrearranged human TRBJ2-4 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ2-3 segment and a mouse TRBJ2-4 segment,                 -   an endogenous TRBJ2-5 gene segment with an                     unrearranged human TRBJ2-5 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ2-4 segment and a mouse TRBJ2-5 segment,                 -   an endogenous TRBJ2-6 gene segment with an                     unrearranged human TRBJ2-6 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ2-5 segment and a mouse TRBJ2-6 segment, and                 -   an endogenous TRBJ2-7 gene segment with an                     unrearranged human TRBJ2-7 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ2-6 segment and a mouse TRBJ2-7 segment or             -   (d) a combination thereof, or         -   (III) the endogenous mouse TCRα variable gene locus             comprises             -   (a) a replacement of all endogenous T cell variable                 region Vα gene segment with all unrearranged human T                 cell variable region Vα gene segments from TRAV1-1 to                 TRAV41,             -   (b) a replacement of all endogenous T cell variable                 region Jα gene segments with all unrearranged human T                 cell variable region Jα gene segments from TRAJ1 to                 TRAJ61, or             -   (c) a combination thereof, and         -   the endogenous mouse TCRβ variable gene locus comprises             -   (a) a replacement of all contiguous endogenous T cell                 variable region Vβ gene segments with all unrearranged                 human T cell variable region Vβ gene segments from TRBV1                 to TRBV29-1,             -   (b) a replacement of endogenous T cell variable region                 Dβ1 gene segment with unrearranged human T cell variable                 region Dβ1 gene segment and a replacement of endogenous                 T cell variable region Dβ2 gene segment with                 unrearranged human T cell variable region Dβ2 gene                 segment,             -   (c) a replacement of                 -   an endogenous TRBJ1-1 gene segment with an                     unrearranged human TRBJ1-1 gene segment,                 -   an endogenous TRBJ1-2 gene segment with an                     unrearranged human TRBJ1-2 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ1-1 segment and a mouse TRBJ1-2 segment,                 -   an endogenous TRBJ1-3 gene segment with an                     unrearranged human TRBJ1-3 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ1-2 segment and a mouse TRBJ1-3 segment,                 -   an endogenous TRBJ1-4 gene segment with an                     unrearranged human TRBJ1-4 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ1-3 segment and a mouse TRBJ1-4 segment,                 -   an endogenous TRBJ1-5 gene segment with an                     unrearranged human TRBJ1-5 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ1-4 segment and a mouse TRBJ1-5 segment,                 -   an endogenous TRBJ1-6 gene segment with an                     unrearranged human TRBJ1-6 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ1-5 segment and a mouse TRBJ1-6 segment,                 -   an endogenous TRBJ2-1 gene segment with an                     unrearranged human TRBJ2-1 gene segment,                 -   an endogenous TRBJ2-2 gene segment with an                     unrearranged human TRBJ2-2 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ2-1 segment and a mouse TRBJ2-2 segment,                 -   an endogenous TRBJ2-3 gene segment with an                     unrearranged human TRBJ2-3 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ2-2 segment and a mouse TRBJ2-3 segment,                 -   an endogenous TRBJ2-4 gene segment with an                     unrearranged human TRBJ2-4 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ2-3 segment and a mouse TRBJ2-4 segment,                 -   an endogenous TRBJ2-5 gene segment with an                     unrearranged human TRBJ2-5 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ2-4 segment and a mouse TRBJ2-5 segment,                 -   an endogenous TRBJ2-6 gene segment with an                     unrearranged human TRBJ2-6 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ2-5 segment and a mouse TRBJ2-6 segment, and                 -   an endogenous TRBJ2-7 gene segment with an                     unrearranged human TRBJ2-7 gene segment, optionally                     wherein the mouse TCRB non-coding sequence comprises                     a mouse non-coding sequence found between a mouse                     TRBJ2-6 segment and a mouse TRBJ2-7 segment, or             -   (d) a combination thereof.     -   Embodiment 7. The mouse or isolated mouse cell of any one of the         preceding embodiments, wherein         -   (I) the endogenous mouse TCRα variable gene locus comprises:             -   (a) a replacement of all endogenous T cell variable                 region Vα gene segments with all unrearranged human T                 cell variable region Vα gene segments from TRAV1-1 to                 TRAV41, and             -   (b) a replacement of all endogenous T cell variable                 region Jα gene segments with all unrearranged human T                 cell variable region Jα gene segments from TRAJ1 to                 TRAJ61; and         -   (II) the endogenous mouse TCRβ variable gene locus comprises             -   (a) a replacement of all contiguous endogenous T cell                 variable region Vβ gene segments with all unrearranged                 human T cell variable region Vβ gene segments from TRBV1                 to TRBV29-1,             -   (b)(i) a replacement of an endogenous tcrbdj1 cluster                 with a humanized TCRBDJ1 cluster comprising an                 unrearranged human TCRBD1 segment and (ii) each of an                 unrearranged human TCRBJ1-1 segment, an unrearranged                 human TCRBJ1-2 segment, an unrearranged human TCRBJ1-3                 segment, an unrearranged human TCRBJ1-4 segment, an                 unrearranged human TCRBJ1-5 segment, and an unrearranged                 human TCRBJ1-6 segment, wherein the humanized TCRBDJ1                 cluster comprises a mouse TCRBDJ1 non-coding sequence                 between the             -   unrearranged human TCRBD1 segment and the unrearranged                 human TCRBJ1-1 segment and a mouse TCRBDJ1 non-coding                 sequence between any two consecutive unrearranged human                 TCRBJ1 gene segments, optionally wherein the                 unrearranged human TCRBD1 and TCRBJ1 gene segments flank                 the same mouse TCRBDJ1 non-coding sequences as are                 normally flanked by the corresponding mouse tcrbdj1 gene                 segments, and             -   (c)(i) a replacement of an endogenous tcrbdj2 cluster a                 humanized TCRBDJ2 cluster comprising an unrearranged                 human TCRBD2 segment and (ii) each of an unrearranged                 human TRBJ2-1 segment, an unrearranged human TRBJ2-2                 segment, an unrearranged human TCRBJ2-3 segment, an                 unrearranged human TCRBJ2-4 segment, an unrearranged                 human TCRBJ2-5 segment, an unrearranged human TCRBJ2-6                 segment, and an unrearranged human TCRBJ2-7 segment,             -   wherein the humanized TCRBDJ2 cluster comprises a mouse                 TCRBDJ2 non-coding sequence between the unrearranged                 human TCRBD2 segment and any unrearranged human TCRBJ2                 segment and a mouse TCRBDJ2 non-coding sequence between                 any two consecutive unrearranged human TCRBJ2 gene                 segments, optionally wherein the unrearranged human                 TCRBD2 and TCRBJ2 gene segments flank the same mouse                 TCRBDJ2 non-coding sequences as are normally flanked by                 the corresponding mouse tcrbdj2 gene segments.     -   Embodiment 8. The mouse or isolated mouse cell of any one of the         preceding embodiments, wherein the unrearranged TCR α variable         region sequence is in the germline genome and/or the         unrearranged TCR β variable region sequence is in the germline         genome.     -   Embodiment 9. The mouse or isolated mouse cell of any one of the         preceding embodiments, wherein the unrearranged TCR α variable         region sequence is expressed under (e.g., operably linked to)         regulatory control of endogenous TCRα regulatory elements and/or         the unrearranged TCR β variable region sequence is expressed         under (e.g., operably linked to) regulatory control of         endogenous TCRβ regulatory elements.     -   Embodiment 10. The mouse or isolated mouse cell of any one of         the preceding embodiments, wherein the mouse or isolated mouse         cell expresses a T cell receptor on the surface of a T cell, the         T cell receptor comprising a humanized TCRα chain and a         humanized TCRβ chain,         -   wherein the humanized TCRα chain is encoded by a rearranged             human Vα/Jα sequence operably linked to the mouse TCRα             constant region sequence, wherein the rearranged human Vα/Jα             sequence is formed by rearrangement of the at least one             human T cell variable region Vα segment and at least one             human T cell variable region Jα segment,         -   wherein the humanized TCRβ chain is encoded by a rearranged             human Vβ/Dβ/Jβ sequence operably linked to the mouse TCRβ             constant region sequence, wherein the rearranged human             Vβ/Dβ/Jβ sequence is formed by rearrangement of the at least             one human T cell variable region Vβ segment, at least one T             cell variable region Dβ segment, and at least one human T             cell variable region Jβ segment, optionally wherein the TCRβ             chain is encoded by a rearranged Vβ/Dβ2/Jβ2 sequence.

Embodiment 11. The mouse or isolated mouse cell of any one of the preceding embodiments, wherein at least 10% of the TCR expressed by the mouse is derived from gene segments from the TCRBDJ1 cluster and at least 10% of the TCR expressed by the mouse is derived from gene segments from the TCRBDJ2 cluster.

-   -   Embodiment 12. A mouse or isolated mouse cell comprising in its         genome         -   (a) a first nucleotide sequence encoding a chimeric             human/mouse CD4 co-receptor that comprises D1, D2 and D3             domains of a human CD4 polypeptide and transmembrane and             cytoplasmic domains of a mouse CD4 polypeptide;         -   (b) a second nucleotide sequence encoding a chimeric             human/mouse CD8α polypeptide and a third nucleotide sequence             encoding a chimeric human/mouse CD8β polypeptide,         -   wherein the chimeric human/mouse CD8α polypeptide comprises             an IgV-like domain of a human CD8α polypeptide and             transmembrane and cytoplasmic domains of a mouse CD8α             polypeptide,         -   wherein the chimeric human/mouse CD8β polypeptide comprises             an IgV-like domain of a human CD8β polypeptide and             transmembrane and cytoplasmic domains of a mouse CD8β             polypeptide;         -   (c) a first nucleic acid sequence encoding a chimeric             human/mouse MHC II α polypeptide and a second nucleic acid             sequence encoding a chimeric human/mouse MHC II β             polypeptide,         -   wherein the chimeric human/mouse MHC II α polypeptide             comprises α1 and α2 domains of a human HLA class II α             polypeptide and transmembrane and cytoplasmic domains of a             mouse MHC II α polypeptide,         -   wherein the chimeric human/mouse MHC II β polypeptide             comprises β1 and β2 domains of a human HLA class II β             polypeptide and transmembrane and cytoplasmic domains of a             mouse MHC II β polypeptide;         -   (d) a third nucleic acid sequence encoding a chimeric             human/mouse MHC I polypeptide,         -   wherein the chimeric MHC I polypeptide comprises α1, α2, and             α3 domains of a human HLA class I polypeptide and             transmembrane and cytoplasmic domains of a mouse MHC I             polypeptide; and         -   (e) an unrearranged human TCR α variable region sequence             comprising at least one human Vα segment and at least one             human Jα segment operably linked to a mouse TCRα constant             region sequence; and an unrearranged TCRβ variable region             sequence comprising the at least one human Vβ segment, the             at least one human Dβ segment, and the at least one human Jβ             segment operably linked to a mouse TCRβ constant region             sequence, wherein the unrearranged TCRβ variable region             sequence comprises a mouse TCRB non-coding sequence,         -   optionally wherein the mouse expresses:         -   (A) the chimeric human/mouse CD4 co-receptor,         -   (B) a chimeric CD8 co-receptor comprising the chimeric             human/mouse CD8α polypeptide and the chimeric human/mouse             CD8β polypeptide,         -   (C) a chimeric MHC II complex comprising the chimeric             human/mouse MHC II α polypeptide and the chimeric             human/mouse MHC II β polypeptide, wherein the chimeric MHC             II complex is capable of binding the chimeric CD4             human/mouse co-receptor, and         -   (D) the chimeric human/mouse MHC I polypeptide, wherein the             chimeric MHC I polypeptide is capable of binding the             chimeric CD8 co-receptor, and         -   (E) a T cell receptor on the surface of a T cell, the T cell             receptor comprising a humanized TCRα chain and a humanized             TCRβ chain,         -   wherein the humanized TCRα chain is encoded by a rearranged             human Vα/Jα sequence operably linked to the mouse TCRα             constant region sequence, wherein the rearranged human Vα/Jα             sequence is formed by rearrangement of the unrearranged             human TCR α variable region sequence comprising at least one             human Vα segment and at least one human Jα segment,         -   wherein the humanized TCRβ chain is encoded by a rearranged             human VR/Dβ/Jβ sequence operably linked to the mouse TCRβ             constant region sequence, wherein the rearranged human             VR/Dβ/Jβ sequence is formed by rearrangement of the             unrearranged TCRβ variable region sequence comprising at             least one human Vβ segment, at least one Dβ segment, and at             least one human Jβ segment.     -   Embodiment 13. The mouse or isolated mouse cell of embodiment         12, comprising in its germline genome         -   (a) the first nucleotide sequence encoding the chimeric             human/mouse CD4 co-receptor;         -   (b) the second nucleotide sequence encoding the chimeric             human/mouse CD8α polypeptide and the third nucleotide             sequence encoding the chimeric human/mouse CD8β polypeptide;         -   (c) the first nucleic acid sequence encoding the chimeric             human/mouse MHC II α polypeptide and the second nucleic acid             sequence encoding the chimeric human/mouse MHC II β             polypeptide;         -   (d) the third nucleic acid sequence encoding the chimeric             human/mouse MHC I polypeptide; and         -   (e) the unrearranged human TCR α variable region sequence             operably linked to a mouse TCRα constant region sequence and             the unrearranged TCRβ variable region sequence operably             linked to a mouse TCRβ constant region sequence.     -   Embodiment 14. The mouse or isolated mouse cell of embodiment 12         or embodiment 13, wherein         -   (a) the first nucleotide sequence is present at an             endogenous CD4 T cell co-receptor locus;         -   (b) the second nucleotide sequence is present at an             endogenous CD8α T cell co-receptor locus and the third             nucleotide sequence is present at an endogenous CD8β T cell             co-receptor locus;         -   (c) the first nucleic acid sequence is present at an             endogenous MHC II α locus and the second nucleic acid             sequence is present at an endogenous MHC II β locus;         -   (d) the third nucleic acid sequence is present at an             endogenous MHC I locus; or         -   (e) the unrearranged human TCRα variable region sequence is             present at an endogenous TCRα variable region locus and the             unrearranged TCRβ variable region sequence is present at an             endogenous TCRβ variable region locus, or         -   (f) any combination of (a)-(e).     -   Embodiment 15. The mouse or isolated mouse cell of any one of         embodiments 12-14, wherein         -   (a) the first nucleotide sequence is present at an             endogenous CD4 T cell co-receptor locus and expressed under             (e.g., operably linked to) regulatory control of endogenous             CD4 co-receptor promoter and regulatory elements;         -   (b) the second nucleotide sequence is present at an             endogenous CD8α T cell co-receptor locus and expressed under             (e.g., operably linked to) regulatory control of endogenous             CD8α polypeptide promoter and regulatory elements, and the             third nucleotide sequence is present at an endogenous CD8β T             cell co-receptor locus and expressed under (e.g., operably             linked to) regulatory control of endogenous CD8β polypeptide             promoter and regulatory elements;         -   (c) the first nucleic acid sequence is present at an             endogenous MHC II α locus and expressed under (e.g.,             operably linked to) regulatory control of endogenous MHC II             α promoter and regulatory elements, and the second nucleic             acid sequence is present at an endogenous MHC II β locus and             expressed under (e.g., operably linked to) regulatory             control of endogenous MHC II β promoter and regulatory             elements;         -   (d) the third nucleic acid sequence is present at an             endogenous MHC I locus and expressed under (e.g., operably             linked to) regulatory control of endogenous MHC I promoter             and regulatory elements;         -   (e) the unrearranged human TCRα variable region sequence is             present at an endogenous TCRα variable region locus and             expressed under (e.g., operably linked to) regulatory             control of endogenous TCRα regulatory elements, and the             unrearranged TCRβ variable region sequence is present at an             endogenous TCRβ variable region locus and expressed under             (e.g., operably linked to) regulatory control of endogenous             TCRβ regulatory elements; or         -   (f) any combination of (a)-(e).     -   Embodiment 16. The mouse or isolated mouse cell of any one of         embodiments 12-15, wherein         -   (a) the chimeric human/mouse CD4 co-receptor comprises D1,             D2 and D3 domains of the human CD4 polypeptide operably             linked to D4, transmembrane, and cytoplasmic domains of an             endogenous CD4 polypeptide,         -   (b) the chimeric human/mouse CD8α polypeptide comprises the             IgV-like domain of the human CD8α polypeptide operably             linked to transmembrane and cytoplasmic domains of an             endogenous CD8α polypeptide and the chimeric CD8β             polypeptide comprises the IgV-like domain of the human CD8β             polypeptide operably linked to transmembrane and cytoplasmic             domains of an endogenous CD8β polypeptide;         -   (c) the chimeric human/mouse MHC II α polypeptide comprises             human HLA class II α1 and α2 domains operably linked to             transmembrane and cytoplasmic domains of an endogenous MHC             II α polypeptide and the chimeric MHC II β polypeptide             comprises human HLA class II β1 and β2 domains operably             linked to transmembrane and cytoplasmic domains of an             endogenous MHC II β polypeptide;         -   (d) the chimeric human/mouse MHC I polypeptide comprises             human HLA class I α1, α2, and α3 domains operably linked             transmembrane and cytoplasmic domains of an endogenous MHC I             polypeptide, or         -   (e) any combination of (a)-(d).     -   Embodiment 17. The mouse or isolated mouse cell of any one of         embodiments 12-16, wherein         -   (a) the α1 and α2 domains of the human HLA class II α             polypeptide are encoded by an HLA class II gene selected             from the group consisting of any a chain gene of HLA-DR,             HLA-DQ, and HLA-DP;         -   (b) the β1 and δ2 domains of the human HLA class II β             polypeptide are encoded by an HLA class II gene selected             from the group consisting of any chain gene of HLA-DR,             HLA-DQ, and HLA-DP;         -   (c) the α1, α2, and α3 domains of the human HLA class I             polypeptide are encoded by a human HLA-A gene, a human HLA-B             gene, or a human HLA-C gene; or         -   (d) any combination of (a)-(c).     -   Embodiment 18. The mouse or isolated mouse cell of any one of         embodiments 12-17, wherein the α1 and α2 domains of the human         HLA class II α polypeptide are encoded by the α chain gene of         HLA-DR and the β1 and δ2 domains of the human HLA class II β         polypeptide are encoded by the β chain gene of HLA-DR, and         -   wherein the α1, α2, and α3 domains of the human HLA class I             polypeptide are encoded by a human HLA-A gene.     -   Embodiment 19. The mouse or isolated mouse cell of any one of         embodiments 12-18, wherein the chimeric MHC II complex comprises         α1, α2, β1, and β2 domains of a human HLA-DR2 protein.     -   Embodiment 20. The mouse or isolated mouse cell of any one of         embodiments 12-19, wherein the chimeric human/mouse MHC I         polypeptide comprises the α1, α2, and α3 domains of a human         HLA-A2 protein.     -   Embodiment 21. The mouse or isolated mouse cell of any one of         embodiments 12-20, wherein         -   (a) the unrearranged human TCRα variable region sequence             comprises a complete repertoire of human Vα gene segments             and a complete repertoire of human Jα gene segments,         -   (b) the unrearranged TCRβ variable region sequence comprises             a complete repertoire of human Vβ gene segments, a complete             repertoire of human Dβ gene segments and a complete             repertoire of human Jβ gene segments, or         -   (c) the unrearranged human TCRα variable region sequence             comprises a complete repertoire of human Vα gene segments             and a complete repertoire of human Jα gene segments and the             unrearranged TCRβ variable region sequences comprises a             complete repertoire of human Vβ gene segments, a complete             repertoire of human Dβ gene segments and a complete             repertoire of human Jβ gene segments.     -   Embodiment 22. The mouse or isolated mouse cell of any one of         embodiments 12-21, wherein:         -   (i) an endogenous TCRα variable locus (a) lacks all or             substantially all functional endogenous Vα gene             segments, (b) lacks all or substantially all functional             endogenous Jα gene segments, or (c) lacks all or             substantially all functional endogenous Vα gene segments and             lacks all or substantially all functional endogenous Jα gene             segments;         -   (ii) an endogenous TCRβ variable locus (a) lacks all or             substantially all functional endogenous Vβ gene             segments, (b) lacks all or substantially all functional             endogenous Dβ gene segments, (c) lacks all or substantially             all functional endogenous Jβ gene segments, or (d) any             combination of (a), (b), and (c); or         -   (iii) both (i) and (ii).     -   Embodiment 23. The mouse or isolated mouse cell of any one of         embodiments 12-22, wherein         -   (a) the first nucleotide sequence comprises a sequence             encoding the D1, D2 and D3 domains of the human CD4             polypeptide that, at an endogenous mouse CD4 locus, (i)             replaces a sequence encoding the D1, D2 and D3 domains of an             endogenous CD4 co-receptor polypeptide and (ii) is operably             linked to endogenous CD4 D4, transmembrane and cytoplasmic             domains encoding sequences;         -   (b) the second nucleotide sequence comprises a sequence             encoding the IgV-like domain of the human CD8α polypeptide             that, at an endogenous CD8α locus, (i) replaces a sequence             encoding an IgV-like domain of the endogenous CD8α             polypeptide and (ii) is operably linked to endogenous CD8α             transmembrane and cytoplasmic domain encoding sequences, and         -   the third nucleotide sequence comprises a sequence encoding             IgV-like domain of the human CD8β polypeptide that, at an             endogenous CD8β locus, (i) replaces a sequence encoding an             IgV-like domain of an endogenous CD8β polypeptide and (ii)             is operably linked to endogenous CD8β transmembrane and             cytoplasmic domain encoding sequences;         -   (c) the first nucleic acid sequence comprises a sequence             encoding the α1 and α2 domains of the human HLA class II α             polypeptide that, at and endogenous MHC II α locus, (i)             replaces a sequence encoding α1 and α2 domains of an             endogenous MHC II α polypeptide and (ii) is operably linked             to endogenous MHC II α polypeptide transmembrane and             cytoplasmic domain encoding sequences, and         -   the second nucleic acid comprises a sequence encoding the β1             and β2 domains of the human HLA class II β polypeptide that,             at an endogenous MHC II β locus, (i) replaces a sequence             encoding β1 and β2 domains of an endogenous MHC II β             polypeptide and (ii) is operably linked to endogenous MHC II             β polypeptide transmembrane and cytoplasmic domain encoding             sequences;         -   (d) the third nucleic acid sequence comprises a sequence             encoding the α1, α2, and α3 domains of the human HLA class I             polypeptide that, at an endogenous MHC I locus, that (i)             replaces a sequence encoding α1, α2, and α3 domains of an             endogenous MHC I polypeptide and (ii) is operably linked to             endogenous MHC I polypeptide transmembrane and cytoplasmic             domain encoding sequences;         -   (e) the unrearranged human TCRα variable region sequence             replaces one or more endogenous Vα and/or Jα gene segments             at an endogenous TCRα variable region locus and the             unrearranged TCRβ variable region sequence replaces one or             more endogenous Vβ, Dβ and/or Jβ gene segments at an             endogenous TCRβ variable region locus; or         -   (f) any combination of (a)-(e).     -   Embodiment 24. The mouse or isolated mouse cell of any one of         embodiments 12-23, wherein the mouse or mouse cell does not         express:         -   (a) a functional endogenous CD4 co-receptor from an             endogenous CD4 co-receptor locus;         -   (b) a functional endogenous CD8 co-receptor from endogenous             CD8 co-receptor loci;         -   (c) an endogenous TCRα variable domain from an endogenous             TCRα locus;         -   (d) an endogenous TCRβ variable domain from an endogenous             TCRβ locus;         -   (e) on a cell surface, an extracellular domain of an             endogenous classical MHC class I polypeptide from an             endogenous MHC I locus;         -   (f) on the surface of a cell, an extracellular domain of an             endogenous classical MHC class II polypeptide from an             endogenous MHC II locus; or         -   (e) any combination of (a)-(f).     -   Embodiment 25. The mouse or isolated mouse cell of any one of         embodiments 12-24, further comprising a β2 microglobulin locus         comprising a sequence encoding a polypeptide comprising a human         β2 microglobulin amino acid sequence, wherein the mouse or mouse         cell expresses a human or humanized β2 microglobulin         polypeptide.     -   Embodiment 26. The mouse or isolated mouse cell of embodiment         25, wherein the mouse or mouse cell does not express a         functional endogenous mouse β2 microglobulin polypeptide from an         endogenous mouse β2 microglobulin locus.     -   Embodiment 27. The mouse or isolated mouse cell of embodiment 25         or embodiment 26, wherein the sequence encoding a polypeptide         comprising a human β2 microglobulin polypeptide is operably         linked to endogenous mouse β2 microglobulin regulatory elements.     -   Embodiment 28. The mouse or isolated mouse cell of any one of         embodiments 25-27, wherein the β2 microglobulin locus comprises         a nucleotide sequence set forth in exon 2, exon 3, and exon 4 of         a human β2 microglobulin gene.     -   Embodiment 29. The mouse or isolated mouse cell of embodiment         28, wherein the β2 microglobulin locus further comprises a         nucleotide sequence set forth in exon 1 of a mouse β2         microglobulin gene.     -   Embodiment 30. A genetically modified mouse or isolated mouse         cell comprising in its genome:         -   (a) a first nucleotide sequence encoding a chimeric             human/mouse CD4 co-receptor that comprises D1, D2 and D3             domains of a human CD4 polypeptide operably linked to D4,             transmembrane and cytoplasmic domains of a mouse CD4             polypeptide;         -   (b) a second nucleotide sequence encoding a chimeric             human/mouse CD8α polypeptide and a third nucleotide sequence             encoding a chimeric human/mouse CD8β polypeptide,         -   wherein the chimeric human/mouse CD8α polypeptide comprises             an IgV-like domain of a human CD8α polypeptide operably             linked to transmembrane and cytoplasmic domains of an             endogenous mouse CD8α polypeptide and wherein the chimeric             human/mouse CD8β polypeptide comprises an IgV-like domain of             a human CD8β polypeptide operably linked to transmembrane             and cytoplasmic domains of an endogenous mouse CD8β             polypeptide;         -   (c) a first nucleic acid sequence encoding a chimeric             human/mouse MHC II α polypeptide and a second nucleic acid             sequence encoding a chimeric human/mouse MHC II β             polypeptide,         -   wherein the chimeric human/mouse MHC II α polypeptide             comprises α1 and α2 domains of a human HLA class II α             polypeptide operably linked to transmembrane and cytoplasmic             domains of an endogenous mouse MHC II α polypeptide and             wherein the chimeric human/mouse MHC II β polypeptide             comprises β1 and β2 domains of a human HLA class II β             polypeptide operably linked to transmembrane and cytoplasmic             domains of an endogenous mouse MHC II β polypeptide;         -   (d) a third nucleic acid sequence encoding a chimeric             human/mouse MHC I polypeptide comprising α1, α2, and α3             domains of a human HLA class I polypeptide operably linked             to transmembrane and cytoplasmic domains of an endogenous             mouse MHC class I polypeptide;         -   (e) an unrearranged human T cell receptor (TCR) α variable             region sequence comprising at least one human Vα segment and             at least one human Jα segment operably linked to a mouse             TCRα constant region sequence; and an unrearranged TCRβ             variable region sequence comprising at least one human Vβ             segment, at least one human Dβ segment, and at least one             human Jβ segment operably linked to a mouse TCRβ constant             region sequence, wherein the unrearranged TCRβ variable             region sequence comprises a mouse TCRB non-coding nucleic             acid sequence; and         -   (f) a polynucleotide encoding a human or humanized β2             microglobulin polypeptide and comprising a nucleotide             sequence comprising the nucleotide sequence set forth in             exon 1 of an endogenous mouse β2 microglobulin gene operably             linked to the nucleotide sequence set forth in exon 2, exon             3, and exon 4 of a human β2 microglobulin gene,         -   optionally wherein the mouse expresses:         -   (A) the chimeric human/mouse CD4 co-receptor,         -   (B) a chimeric CD8 co-receptor comprising the chimeric             human/mouse CD8α polypeptide and the chimeric human/mouse             CD8β polypeptide,         -   (C) a chimeric MHC II complex comprising the chimeric             human/mouse MHC II α polypeptide and the chimeric             human/mouse MHC II β polypeptide, wherein the chimeric MHC             II complex is capable of binding the chimeric human/mouse             CD4 co-receptor,         -   (D) the chimeric human/mouse MHC I polypeptide, wherein the             chimeric MHC I polypeptide is capable of binding the             chimeric CD8 co-receptor,         -   (E) a chimeric human/mouse T cell receptor comprising a             humanized TCRα chain and a humanized TCRβ chain, on the             surface of a T cell,         -   wherein the humanized TCRα chain is encoded by a rearranged             human Vα/Jα sequence operably linked to the mouse TCRα             constant region sequence, wherein the rearranged human Vα/Jα             sequence is formed by rearrangement of the unrearranged             human TCR α variable region sequence comprising the at least             one human Vα segment and the at least one human Jα segment,         -   wherein the humanized TCRβ chain is encoded by a rearranged             human Vβ/Dβ/Jβ sequence operably linked to the mouse TCRβ             constant region sequence, wherein the rearranged human             Vβ/Dβ/Jβ sequence is formed by rearrangement of the             unrearranged human TCR β variable region comprising the at             least one human Vβ segment, at least one Dβ segment, and at             least one human Jβ segment, optionally wherein the humanized             TCRβ chain is encoded by a rearranged human Vβ/Dβ2/Jβ2             sequence operably linked to the mouse TCRβ constant region             sequence, and         -   (F) the human or humanized β2 microglobulin polypeptide.     -   Embodiment 31. The mouse of any one of embodiments 12-30,         wherein at least 10% of the TCR expressed by the mouse is         derived from gene segments from the TCRBDJ1 cluster and at least         10% of the TCR expressed by the mouse is derived from gene         segments from the TCRBDJ2 cluster.     -   Embodiment 32. The mouse or mouse cell of any one of embodiments         12-31, wherein the chimeric human/mouse MHC II α polypeptide is         a chimeric HLA-DR/H-2Eα polypeptide, the chimeric human/mouse         MHC II β polypeptide is a chimeric human/mouse HLA-DR/H-2E β         polypeptide, and the chimeric human/mouse MHC I polypeptide is a         chimeric human/mouse HLA-A/H-2K polypeptide, and wherein the         mouse expresses HLA-A/H-2K and HLA-DR/H-2E proteins.     -   Embodiment 33. A method of making the mouse or mouse cell of any         one of embodiments 1-32, comprising modifying the genome of the         mouse or mouse cell to comprise         -   (a) the unrearranged T cell receptor (TCR) α variable region             sequence comprising at least one unrearranged human T cell             variable region Vα segment and at least one unrearranged             human T cell variable region Jα segment operably linked to a             mouse TCR α constant gene sequence, optionally at an             endogenous mouse TCR α variable gene locus, wherein the             unrearranged TCR α variable region sequence comprises a             mouse TCRA non-coding sequence,         -   (b) the unrearranged TCRβ variable region sequence             comprising at least one unrearranged human T cell variable             region Vβ segment, at least one unrearranged human T cell             variable region Dβ segment, and at least one unrearranged             human T cell variable region Jβ segment operably linked to a             mouse TCRβ constant gene sequence, optionally at an             endogenous mouse TCRβ variable gene locus,         -   wherein the unrearranged TCRβ variable region sequence             comprises a mouse TCRB non-coding sequence, or         -   (c) both the unrearranged TCR α variable region sequence and             the unrearranged TCRβ variable region sequence.     -   Embodiment 34. The method of embodiment 33, wherein the method         comprises replacing a contiguous mouse TCRB sequence comprising         a mouse TCRBD gene segment and a mouse TCRBJ gene segment with a         nucleic acid sequence comprising the at least one unrearranged         human T cell variable region DR segment, a mouse TCRBD-TCRBJ         non-coding nucleic acid sequence, and the at least one         unrearranged human T cell variable region Jβ segment, such that         the at least one unrearranged human T cell variable region Dβ         segment, the mouse TCRBD-TCRBJ non-coding nucleic acid sequence,         and the at least one unrearranged human T cell variable region         Jβ segment are operably linked to the mouse TCRβ constant gene         sequence.     -   Embodiment 35. The method of embodiment 34, wherein the         contiguous mouse TCRB sequence comprises (a) a mouse TCRBD1 gene         segment and a mouse TCRBJ1-6 gene segment and/or (b) a mouse         TCRBD2 gene segment and a mouse TCRBJ2-7 gene segment, and         -   wherein the nucleic acid comprises:         -   (A) a humanized TCRBDJ1 cluster comprising an unrearranged             human TCRBD1 gene segment, an unrearranged human TCRBJ1 gene             segment, and a mouse TCRBDJ1 non-coding sequence, wherein             the humanized TCRBDJ1 cluster comprises:             -   an unrearranged human TRBJ1-1 gene segment, an                 unrearranged human TRBJ1-2 gene segment, and a mouse                 TCRB non-coding sequence between the unrearranged human                 TRBJ1-1 gene segment and the unrearranged human TRBJ1-2                 gene segment,             -   an unrearranged human TRBJ1-2 gene segment, an                 unrearranged human TRBJ1-3 gene segment, and a mouse                 TCRB non-coding sequence between the unrearranged human                 TRBJ1-2 gene segment and the unrearranged human TRBJ1-3                 gene segment,             -   an unrearranged human TRBJ1-3 gene segment, an                 unrearranged human TRBJ1-4 gene segment, and a mouse                 TCRB non-coding sequence between the unrearranged human                 TRBJ1-3 gene segment and the unrearranged human TRBJ1-4                 gene segment,         -   an unrearranged human TRBJ1-4 gene segment, an unrearranged             human TRBJ1-5 gene segment, and a mouse TCRB non-coding             sequence between the unrearranged human TRBJ1-4 gene segment             and the unrearranged human TRBJ1-5 gene segment,         -   an unrearranged human TRBJ1-5 gene segment, an unrearranged             human TRBJ1-6 gene segment, and a mouse TCRB non-coding             sequence between the unrearranged human TRBJ1-5 gene segment             and the unrearranged human TRBJ1-6 gene segment, or             -   any combination of an unrearranged human TRBJ1-1 gene                 segment, an unrearranged human TRBJ1-2 gene segment, an                 unrearranged human TRBJ1-3 gene segment, an unrearranged                 human TRBJ1-4 gene segment, an unrearranged human                 TRBJ1-5 gene segment, and an unrearranged human TRBJ1-6                 gene segment, wherein the humanized TCRBDJ1 cluster                 comprises a mouse TCRBDJ1 non-coding sequence between                 the unrearranged human TCRBD1 gene segment and any                 unrearranged human TCRBJ1 gene segment and mouse TCRBDJ1                 non-coding sequence between any two consecutive                 unrearranged human TCRBJ1 gene segments, optionally                 wherein the unrearranged human TCRBD1 and TCRBJ1 gene                 segments flank the same mouse TCRBDJ1 non-coding                 sequences as are normally flanked by the corresponding                 mouse tcrbdj1 gene segments; and/or         -   (B) a humanized TCRBDJ2 cluster comprising an unrearranged             human TCRBD2 gene segment, an unrearranged human TCRBJ2 gene             segment, and a mouse TCRBDJ2 non-coding sequence, wherein             the humanized TCRBDJ2 cluster comprises:             -   an unrearranged human TRBJ2-1 gene segment, an                 unrearranged human TRBJ2-2 gene segment, and a mouse                 TCRB non-coding sequence between the unrearranged human                 TRBJ2-1 gene segment and the             -   an unrearranged human TRBJ2-2 gene segment,             -   unrearranged human TRBJ2-2 gene segment, an unrearranged                 human TRBJ2-3 gene segment, and a mouse TCRB non-coding                 sequence between the unrearranged human TRBJ2-2 gene                 segment and the unrearranged human TRBJ2-3 gene segment,             -   an unrearranged human TRBJ2-3 gene segment, an                 unrearranged human TRBJ2-4 gene segment, and a mouse                 TCRB non-coding sequence between the unrearranged human                 TRBJ2-3 gene segment and the unrearranged human TRBJ2-4                 gene segment,             -   an unrearranged human TRBJ2-4 gene segment, an                 unrearranged human TRBJ2-5 gene segment, and a mouse                 TCRB non-coding sequence between the unrearranged human                 TRBJ2-4 gene segment and the unrearranged human TRBJ2-5                 gene segment,             -   an unrearranged human TRBJ2-5 gene segment, an                 unrearranged human TRBJ2-6 gene segment, and a mouse                 TCRB non-coding sequence between the unrearranged human                 TRBJ2-5 gene segment and the unrearranged human TRBJ2-6                 gene segment,             -   an unrearranged human TRBJ2-6 gene segment, an                 unrearranged human TRBJ2-7 gene segment, and a mouse                 TCRB non-coding sequence between the unrearranged human                 TRBJ2-6 gene segment and the unrearranged human TRBJ27                 gene segment, or             -   any combination of an unrearranged human TRBJ2-1 gene                 segment, an unrearranged human TRBJ2-2 gene segment, an                 unrearranged human TRBJ2-3 gene segment, an unrearranged                 human TRBJ2-4 gene segment, an unrearranged human                 TRBJ2-5 gene segment, an unrearranged human TRBJ2-6 gene                 segment, and an unrearranged human TRBJ2-7 gene segment,                 wherein the humanized TCRBDJ2 cluster comprises a mouse                 TCRBDJ2 non-coding sequence between the unrearranged                 human TCRBD2 gene segment and any unrearranged human                 TCRBJ2 gene segment and mouse TCRBDJ2 non-coding                 sequence between any two consecutive unrearranged human                 TCRBJ2 gene segments, optionally wherein the                 unrearranged human TCRBD2 and TCRBJ2 gene segments flank                 the same mouse TCRBDJ2 non-coding sequences as are                 normally flanked by the corresponding mouse tcrbdj1 gene                 segments.     -   Embodiment 36. The method of any one of embodiments 33-35,         further comprising modifying the genome of the mouse or the         mouse cell to comprise:         -   (a) the first nucleotide sequence encoding the chimeric CD4             co-receptor;         -   (b) the second nucleotide sequence encoding the chimeric             CD8α polypeptide and the third nucleotide sequence encoding             the chimeric CD8β polypeptide;         -   (c) the first nucleic acid sequence encoding the chimeric             MHC II α polypeptide and the second nucleic acid sequence             encoding the chimeric MHC II β polypeptide;         -   (d) the third nucleic acid sequence encoding the chimeric             MHC I polypeptide; and         -   (e) optionally, a β2 microglobulin locus encoding a human or             humanized β2 microglobulin polypeptide.     -   Embodiment 37. The method of embodiment 36, wherein the         modifying the genome comprises homologous recombination in one         or more mouse ES cell(s) such that the first, second, and third         nucleotide sequences; the unrearranged human TCRα variable         region sequence and unrearranged TCRβ variable region sequence;         the first, second, and third nucleic acid sequences; and         optionally the β2 microglobulin locus; are added, in any order,         into the genome of the one or more mouse ES cell(s).     -   Embodiment 38. The method of embodiment 37, further comprising         generating a mouse from the one or more mouse ES cell(s).     -   Embodiment 39. A method of obtaining any one of: (1) a TCR         protein that is specific for an antigen and comprises a human         TCR variable domain, (2) the human TCR variable domain and (3) a         nucleic acid sequence encoding the human TCR variable domain,         -   the method comprising         -   isolating from a mouse according to any one of embodiments             1-32 any one of:             -   (1) a T cell expressing a TCR protein that is specific                 for an antigen and comprises both a human TCR α variable                 domain and a human TCR β variable domain,             -   (2) either or both (i) the human TCR α variable domain                 and (ii) the human TCR β variable domain, and             -   (3) either or both (i) a nucleic acid sequence encoding                 the human TCR α variable domain and (ii) a nucleic acid                 sequence encoding the human TCR β variable domain.     -   Embodiment 40. The method of embodiment 39, wherein the method         comprises isolating from the mouse a nucleic acid sequence         encoding the human TCR α variable domain and a nucleic acid         sequence encoding the human TCR β variable domain, the method         further comprising         -   culturing a host cell in sufficient conditions for             expressing (i) the nucleic acid sequence encoding the human             TCR α variable domain in operable linkage with a human TCR α             constant region (ii) the nucleic acid sequence encoding the             human TCR β variable domain in operable linkage with a human             TCR β constant region,         -   wherein the nucleic acid sequences encoding the human TCR α             variable domain and the human TCR β variable domain are on             the same or different expression vectors.     -   Embodiment 41. The method of embodiment 39 or embodiment 40,         wherein the antigen is a tumor antigen, a viral antigen, or a         bacterial antigen.     -   Embodiment 42. A T cell expressing a TCR protein comprising a         human TCR variable domain specific for an antigen obtained         according to the method of any one of embodiments 39-41.     -   Embodiment 43. A hybridoma produced from the T cell expressing a         TCR protein comprising a human TCR variable domain specific for         an antigen obtained according to the method of any one of         embodiments 39-42.     -   Embodiment 44. A human T cell receptor variable domain obtained         according to the method of any one of embodiments 39-43.     -   Embodiment 45. A nucleic acid comprising either or both (i) the         nucleic acid sequence encoding the human TCR α variable domain         and (ii) the nucleic acid sequence encoding the human TCR β         variable domain isolated according to the method of any one of         embodiments 39-44.     -   Embodiment 46. A host cell comprising the nucleic acid of         embodiment 45.     -   Embodiment 47. An expression vector comprising the nucleic acid         of embodiment 45.     -   Embodiment 48. The expression vector of embodiment 47, wherein         the expression vector comprises at least one of (a) the nucleic         acid sequence encoding a human TCRα variable domain operably         linked to a sequence encoding a human TCRα constant region         and (b) the nucleic acid sequence encoding the human TCRβ         variable domain operably linked to a sequence encoding a human         TCRβ constant region.     -   Embodiment 49. The mouse cell of any one of embodiments 1-32,         wherein the mouse cell is an embryonic stem cell.     -   Embodiment 50. The genetically modified mouse embryonic stem         cell of embodiment 49, comprising in its genome         -   (a) a first nucleotide sequence encoding a chimeric CD4             co-receptor that comprises D1, D2 and D3 domains of a human             CD4 polypeptide and transmembrane and cytoplasmic domains of             a mouse CD4 polypeptide,         -   (b) a second nucleotide sequence and a third nucleotide             sequence respectively encoding a chimeric CD8α polypeptide             and a chimeric CD8β polypeptide,         -   wherein the chimeric CD8α polypeptide comprises an IgV-like             domain of a human CD8α polypeptide and transmembrane and             cytoplasmic domains of a mouse CD8α polypeptide,         -   wherein the chimeric CD8β polypeptide comprises an IgV-like             domain of a human CD8β polypeptide and transmembrane and             cytoplasmic domains of a mouse CD8β polypeptide, and         -   (c) a first nucleic acid sequence and a second nucleic acid             sequence respectively encoding a chimeric MHC II α             polypeptide and a chimeric MHC II β polypeptide,         -   wherein the chimeric MHC II α polypeptide comprises α1 and             α2 domains of a human HLA class II α polypeptide and             transmembrane and cytoplasmic domains of a non-human MHC II             α polypeptide,         -   wherein the chimeric MHC II β polypeptide comprises β1 and             β2 domains of a human HLA class II β polypeptide and             transmembrane and cytoplasmic domains of a mouse MHC II β             polypeptide,         -   (d) a third nucleic acid sequence encoding a chimeric MHC I             polypeptide,         -   wherein the chimeric MHC I polypeptide comprises α1, α2, and             α3 domains of a human HLA class I polypeptide and             transmembrane and cytoplasmic domains of a mouse MHC I             polypeptide, and         -   (e) an unrearranged human T cell receptor (TCR) α variable             region sequence comprising at least one human Vα segment and             at least one human Jα segment operably linked to mouse TCRα             constant region sequence; and an unrearranged TCRβ variable             region sequence comprising at least one human Vβ segment, at             least one human Dβ segment, and at least one human Jβ             segment operably linked to a mouse TCRβ constant region             sequence, wherein the unrearranged TCRβ variable region             sequence comprises a mouse TCRB non-coding sequence.     -   Embodiment 51. The genetically modified mouse embryonic stem         cell of embodiment 50, wherein         -   (a) the first nucleotide sequence is present at an             endogenous CD4 T cell co-receptor locus;         -   (b) the second nucleotide sequence is present at an             endogenous CD8α T cell co-receptor locus, and the third             nucleotide sequence is present at an endogenous CD8β T cell             co-receptor locus;         -   (c) the first nucleic acid sequence is present at an             endogenous MHC II α locus and the second nucleic acid             sequence is present at an endogenous MHC II β locus;         -   (d) the third nucleic acid sequence is present at an             endogenous MHC I locus; and/or         -   (e) the unrearranged human TCRα variable region sequence is             present at an endogenous TCRα variable region locus and the             unrearranged TCRβ variable region sequence is present at an             endogenous TCRβ variable region locus.     -   Embodiment 52. The genetically modified mouse embryonic stem         cell of embodiment 50 or embodiment 51, wherein         -   (a) the first nucleotide sequence is present at an             endogenous CD4 T cell co-receptor locus and operably linked             to endogenous CD4 co-receptor promoter and regulatory             elements;         -   (b) the second nucleotide sequence is present at an             endogenous CD8α T cell co-receptor locus and operably linked             to endogenous CD8α polypeptide promoter and regulatory             elements, and the third nucleotide sequence is present at an             endogenous CD8β locus operably linked to endogenous CD8β             polypeptide promoter and regulatory elements;         -   (c) the first nucleic acid sequence is present at an             endogenous MHC II α locus operably linked to endogenous MHC             II α promoter and regulatory elements and the second nucleic             acid sequence is present at an endogenous MHC II β locus and             operably linked to endogenous non-human MHC II β promoter             and regulatory elements;         -   (d) the third nucleic acid sequence is present at an             endogenous MHC I locus operably linked to endogenous MHC I             promoter and regulatory elements; and/or         -   (e) the unrearranged TCRα variable region sequence is             operably linked to endogenous TCRα regulatory elements and             the unrearranged TCRβ variable region sequence is operably             linked to endogenous TCRβ regulatory elements.     -   Embodiment 53. The genetically modified mouse embryonic stem         cell of any one of embodiments 50-52, wherein the first         nucleotide sequence encodes the chimeric CD4 co-receptor         comprising D1, D2 and D3 domains of the human CD4 polypeptide         operably linked to D4, transmembrane and cytoplasmic domains of         an endogenous CD4 polypeptide,         -   (b) the second nucleotide sequence encodes the chimeric CD8α             polypeptide comprising the IgV-like domain of the human CD8α             polypeptide operably linked to transmembrane and cytoplasmic             domains of an endogenous CD8α polypeptide and the third             nucleotide sequence encodes the chimeric CD8β polypeptide             comprising the IgV-like domain of the human CD8β polypeptide             operably linked to transmembrane and cytoplasmic domains of             an endogenous CD8β polypeptide;         -   (c) the first nucleic acid sequence encodes the chimeric MHC             II α polypeptide comprising human HLA class II α1 and α2             domains operably linked to transmembrane and cytoplasmic             domains of an endogenous MHC II α polypeptide and the second             nucleic acid sequence encodes the chimeric MHC II β             polypeptide comprising human HLA class II β1 and β2 domains             operably linked to transmembrane and cytoplasmic domains of             an endogenous MHC II β polypeptide; and/or         -   (d) the third nucleic acid sequence encodes the chimeric MHC             I polypeptide comprising human HLA class I α1, α2, and α3             domains operably linked to transmembrane and cytoplasmic             domains of an endogenous MHC I polypeptide.     -   Embodiment 54. A mouse embryonic stem (ES) cell made by the         method of any one of embodiments 33-38.     -   Embodiment 55. A targeting vector comprising 5′ and 3′ homology         arms for targeting a mouse TCRBDJ region, an unrearranged human         TCRBD segment, an unrearranged human TCRBJ segment, and a mouse         TRCBDJ non-coding sequence,         -   wherein targeting vector comprises the mouse TCRBDJ             non-coding sequence between the unrearranged human TCRBD             segment and any unrearranged human TCRBJ gene segment and             between any two consecutive unrearranged human TCRBJ gene             segments, optionally wherein the unrearranged human TCRBD             and TCRBJ gene segments flank the same mouse TCRBDJ             non-coding sequences as are normally flanked by the             corresponding mouse tcrbdj gene segments.     -   Embodiment 56. The targeting vector of embodiment 55, wherein         -   (A) the unrearranged human TCRBD segment comprises a             sequence set forth at the following human genomic             coordinates on chromosome 7 (GRCh38 assembly):             -   142,786,213-142,786,224, and/or             -   142,796,365-142,796,414;         -   (B) the unrearranged human TCRBJ segment comprises a             sequence set forth at the following human genomic             coordinates on chromosome 7 (GRCh38 assembly):             -   142,786,880-142,786,927,             -   142,787,017-142,787,064,             -   142,787,630-142,787,679,             -   142,788,225-142,788,275,             -   142,788,498-142,788,547,             -   142,788,988-142,789,040,             -   142,795,686-142,795,740,             -   142,796,560-142,796,610,             -   142,796,847-142,796,895,             -   142,796,998-142,797,047,             -   142,797,119-142,797,166,             -   142,797,239-142,797,291, and/or             -   142,797,456-142,797,502.     -   Embodiment 57. A mouse genome or mouse cell comprising the         targeting vector of embodiment 55 or embodiment 56.

Other uses of the genetically modified animals described herein, i.e., animals comprising a human or humanized T cell co-receptor (e.g., chimeric human/non-human CD4 or CD8), optionally further comprising a human or humanized MHC II or I protein, will be apparent from the present disclosure.

EXAMPLES

The following examples are provided so as to describe to those of ordinary skill in the art how to make and use methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. The Examples do not include detailed descriptions of conventional methods that would be well known to those of ordinary skill in the art (molecular cloning techniques, etc.). Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is indicated in Celsius, and pressure is at or near atmospheric.

Example 1: Generation of Humanized MHC Mice

The various steps involved in engineering a mouse comprising humanized MHC I and MHC II loci, with corresponding and additional endogenous MHC I and MHC II loci deletions (HLA-A2/H-2K, HLA-DR2/H-2E, H-2A-del, H-2D-del) are depicted in FIG. 3A. Detailed description of the steps appears below.

Example 1.1: Generation and Characterization of Humanized MHC I Mice

Generation of humanized MHC I mice has previously been described in U.S. Patent Publication No. 20130111617, incorporated herein by reference. Briefly, the mouse H-2K gene was humanized in a single step by construction of a unique targeting vector from human and mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat. Biotech. 21(6): 652-659). DNA from mouse BAC clone RP23-173k21 (Invitrogen) was modified by homologous recombination to replace the genomic DNA encoding the α1, α2 and α3 domains of the mouse H-2K gene with human genomic DNA encoding the α1, α2 and α3 subunits of the human HLA-A gene (FIG. 2A).

Specifically, the genomic sequence encoding the mouse the α1, α2 and α3 subunits of the H-2K gene is replaced with the human genomic DNA encoding the α1, α2 and α3 domains of the human HLA-A*0201 gene in a single targeting event using a targeting vector comprising a hygromycin cassette flanked by loxP sites with a 5′ mouse homology arm containing sequence 5′ of the mouse H-2K locus including the 5′ untranslated region (UTR) and a 3′ mouse homology arm containing genomic sequence 3′ of the mouse H-2K α3 coding sequence.

The final construct for targeting the endogenous H-2K gene locus from 5′ to 3′ included (1) a 5′ homology arm containing ˜200 bp of mouse genomic sequence 5′ of the endogenous H-2K gene including the 5′UTR, (2) ˜1339 bp of human genomic sequence including the HLA-A*0201 leader sequence, the HLA-A*0201 leader/α1 intron, the HLA-A*0201 α1 exon, the HLA-A*0201 α1-α2 intron, the HLA-A*0201 α2 exon, ˜316 bp of the 5′ end of the α2-α3 intron, (3) a 5′ loxP site, (4) a hygromycin cassette, (5) a 3′ loxP site, (6) ˜580 bp of human genomic sequence including ˜304 bp of the 3′ end of the α2-α3 intron, the HLA-A*0201 α3 exon, and (7) a 3′ homology arm containing ˜200 bp of mouse genomic sequence including the intron between the mouse H-2K α3 and transmembrane coding sequences. The sequence of 149 nucleotides at the junction of the mouse/human sequences at the 5′ of the targeting vector is set forth in SEQ ID NO: 90, and the sequence of 159 nucleotides at the junction of the human/mouse sequences at the 3′ of the targeting vector is set forth in SEQ ID NO:91. Homologous recombination with this targeting vector created a modified mouse H-2K locus containing human genomic DNA encoding the α1, α2 and α3 domains of the HLA-A*0201 gene operably linked to the endogenous mouse H-2K transmembrane and cytoplasmic domain coding sequences which, upon translation, leads to the formation of a chimeric human/mouse MHC class I protein. The selection cassette present in the targeting construct may be later removed using various methods known in the art.

The targeted BAC DNA was used to electroporate mouse F1H4 ES cells to create modified ES cells for generating mice that express a chimeric MHC class I protein on the surface of nucleated cells (e.g., T and B lymphocytes, macrophages, neutrophils) (see, e.g., step 1 in the scheme depicted in FIG. 3A). ES cells containing an insertion of human HLA sequences were identified by a quantitative TAQMAN™ assay (Valenzuela et al. (2003), supra).

To generate mice expressing chimeric MHC I, targeted ES cells described herein are used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses Nature Biotech. 25(1):91-99). VELOCIMICE® (F0 mice fully derived from the donor ES cell) independently bearing a chimeric MHC class I gene are identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the unique human HLA-A*0201 gene sequences. Heterozygous mice generated by this method are bred to homozygosity. Expression of chimeric HLA-A2/H-2K is confirmed by flow cytometry using antibodies specific for HLA-A and H-2K.

Targeted ES cells described above comprising the chimeric HLA-A2/H-2K were used in further genetic engineering steps described in Examples 1.2-1.3 to generate mice comprising both humanized MHC I and MHC II loci and lacking endogenous MHC I and MHC II loci (See FIG. 3A).

Example 1.2: Generation of Mouse ES Cells Comprising MHC I and MHC II Loci Deletions

Deletion of endogenous MHC II loci is described in U.S. Patent Application Number No. 20130111616, incorporated herein by reference. Briefly, the targeting vector for introducing a deletion of the endogenous MHC class II H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea genes was made using VELOCIGENE® genetic engineering technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al., supra). Bacterial Artificial Chromosome (BAC) RP23-458i22 (Invitrogen) DNA was modified to delete the endogenous MHC class II genes H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea.

Specifically, upstream and downstream homology arms were derived by PCR of mouse BAC DNA from locations 5′ of the H-2Ab1 gene and 3′ of the H-2Ea gene, respectively. These homology arms were used to make a cassette that deleted ˜79 kb of RP23-458i22 comprising genes H-2Ab1, H-2Aa, H-2Eb1, H-2Eb2, and H-2Ea of the MHC class II locus by bacterial homologous recombination (BHR). This region was replaced with a neomycin cassette flanked by lox2372 sites. The final targeting vector from 5′ to 3′ included a 26 kb homology arm comprising mouse genomic sequence 5′ to the H-2Ab1 gene of the endogenous MHC class II locus, a 5′ lox2372 site, a neomycin cassette, a 3′ lox2372 site and a 63 kb homology arm comprising mouse genomic sequence 3′ to the H-2Ea gene of the endogenous MHC class II locus.

The BAC DNA targeting vector (described above) was used to electroporate mouse ES cells comprising humanized MHC I locus (from Example 1.1 above; see, e.g., step 2 in FIG. 3A) to create modified ES cells comprising a deletion of the endogenous MHC class II locus (both H-2A and H-2E were deleted). Positive ES cells containing a deleted endogenous MHC class II locus were identified by the quantitative PCR assay using TAQMAN™ probes (Lie and Petropoulos (1998) Curr. Opin. Biotechnology 9:43-48). The upstream region of the deleted locus was confirmed by PCR using primers 5111U F (CAGAACGCCAGGCTGTAAC; SEQ ID NO:1) and 5111U R (GGAGAGCAGGGTCAGTCAAC; SEQ ID NO:2) and probe 5111U P (CACCGCCACTCACAGCTCCTTACA; SEQ ID NO:3), whereas the downstream region of the deleted locus was confirmed using primers 5111D F (GTGGGCACCATCTTCATCATTC; SEQ ID NO:4) and 5111D R (CTTCCTTTCCAGGGTGTGACTC; SEQ ID NO:5) and probe 5111D P (AGGCCTGCGATCAGGTGGCACCT; SEQ ID NO:6). The presence of the neomycin cassette from the targeting vector was confirmed using primers NEOF (GGTGGAGAGGCTATTCGGC; SEQ ID NO:7) and NEOR (GAACACGGCGGCATCAG; SEQ ID NO:8) and probe NEOP (TGGGCACAACAGACAATCGGCTG; SEQ ID NO:9). The nucleotide sequence across the upstream deletion point (SEQ ID NO:10) included the following, which indicates endogenous mouse sequence upstream of the deletion point (contained within the parentheses below) linked contiguously to cassette sequence present at the deletion point: (TTTGTAAACA AAGTCTACCC AGAGACAGAT GACAGACTTC AGCTCCAATG CTGATTGGTT CCTCACTTGG GACCAACCCT) ACCGGTATAA CTTCGTATAA GGTATCCTAT ACGAAGTTAT ATGCATGGCC TCCGCGCCGG. The nucleotide sequence across the downstream deletion point (SEQ ID NO:11) included the following, which indicates cassette sequence contiguous with endogenous mouse sequence downstream of the deletion point (contained within the parentheses below): CGACCTGCAG CCGGCGCGCC ATAACTTCGT ATAAGGTATC CTATACGAAG TTATCTCGAG (CACAGGCATT TGGGTGGGCA GGGATGGACG GTGACTGGGA CAATCGGGAT GGAAGAGCAT AGAATGGGAG TTAGGGAAGA).

Subsequently to generation of the ES cells comprising both the MHC I humanization and endogenous MHC II deletion described above, the loxed neomycin cassette was removed using CRE (see, e.g., step 3 in FIG. 3A). Specifically, a plasmid encoding Cre recombinase was electroporated into ES cells to remove the neomycin cassette. Neo cassette may also be removed using other methods known in the art.

To delete mouse H-2D locus, BHR was used to modify mouse BAC clone bMQ-218H21 (Sanger Institute), replacing 3756 bp of the H2-D gene (from the ATG start codon to 3 bp downstream of the TGA stop codon, exons 1-8 of mouse H-2D) with a 6,085 bp cassette containing from 5′ to 3′: a LacZ gene in frame with a 5′ loxp site, UbC promoter, Neomycin gene, and 3′ loxp site.

The BAC DNA targeting vector (described above) was used to electroporate mouse ES cells comprising humanized MHC I locus and a deletion of mouse MHC II, described above (see, e.g., step 4 in FIG. 3A). Positive ES cells containing a deleted endogenous H-2D locus were identified by the quantitative PCR assay, as described above. Table 2 contains primers and probes used for the quantitative PCR assay.

TABLE 2 TAQMAN™ Loss of Allele Assay Primers and Probes for Detection of Deleted H-2D Locus Name Forward Reverse (location) Primer Primer Probe 5152 mTU CGAGGAGC AAGCGCACGA CTCTGTCG (upstream) CCCGGTACA CACTCTTGTT GCTATGTGG (SEQ ID (SEQ ID (SEQ ID NO: 12) NO: 13) NO: 14) 5152 mTD GGACTCCCA GAGTCATGAA TGGTGGGT (downstream) GAATCTCCT CCATCACTGTG TGCTGGAA GAGA AAGA (SEQ ID (SEQ ID (SEQ ID NO: 17) NO: 15) NO: 16)

Example 1.3: Introduction of Chimeric Human/Mouse MHC II Locus

To generate a vector comprising humanized HLA-DR2/H-2E, first, mouse H-2Eα gene was modified in accordance with the description in U.S. Pat. No. 8,847,005, issued Sep. 30, 2014, incorporated herein by reference, to generate a vector comprising sequence encoding a chimeric H-2Ea/HLA-DRA1*01 protein.

For mouse H-2Eb gene, synthesized human HLA-DR2β chain (DRB1*1501) was used to generate a vector comprising DRβ1*02(1501) exons and introns, and swapped using bacterial homologous recombination into the vector comprising chimeric H-2Ea/HLA-DRA1*01 protein. H-2Eb1 gene was modified essentially as described in U.S. Patent Publication No. 20130185820, and U.S. Pat. No. 8,847,005, each incorporated herein by reference. A hygromycin selection cassette was used.

The resulting HLA-DR2/H-2E large targeting vector (LTVEC) is depicted in FIGS. 2B and 3B. The various nucleotide sequence junctions of the resulting LTVECs (e.g., mouse/human sequence junctions, human/mouse sequence junctions, or junctions of mouse or human sequence with selection cassettes) are summarized below in Table 3 and listed in the Sequence Listing; their locations are indicated in the schematic diagram of FIG. 3B. In Table 3 below, with the exception of sequences marked with asterisks (*, see Table legend) the mouse sequences are in regular font; the human sequences are in parentheses; the Lox sequences are italicized; and the restriction sites introduced during cloning steps and other vector-based sequences (e.g., multiple cloning sites, etc.) are bolded.

TABLE 3 Nucleotide Sequence Junctions of Chimeric HLA-DR2/H-2E Locus SEQ ID NO: Nucleotide Sequence  18 CTGTTTCTTC CCTAACTCCC ATTCTATGCT CTTCCATCCC GA CCGCGG(CCCA ATCTCTCTCC ACTACTTCCT GCCTACATGT ATGTAGGT) 19 (CAAGGTTTCC TCCTATGATG CTTGTGTGAA ACTCGG) GGCC GGCC AGCATTTAAC AGTACAGGGA TGGGAGCACA GCTCAC 20* (GAAAGCAGTC TTCCCAGCCT TCACACTCAG AGGTACAAAT) CCCCATTTTC ATATTAGCGA TTTTAATTTA TTCTAGCCTC 21* TCTTCCCTAA CTCCCATTCT ATGCTCTTCC ATCCCGA CCG CGG(CCCAATC TCTCTCCACT ACTTCCTGCC TACATGTATG) 22 GAGTTCCTCCATCACTTCACTGGGTAGCACA GCTGTAACTGTCCAGCCTG (TCCTGGGCTGCAGGTGGTGGGCGTTGCGGG TGGGGCCGGTTAAGGTTCCA) 23 (TCCCACATCCTATTTTAATTTGCTCCATGT TCTCATCTCCATCAGCACAG) CTCGAG ATAACTTCGTATAATGTATGCTA TACGAAGTTAT ATGCATGGCC 24 ATACGAAGTTAT GCTAGTAACTATAACGG TCCTAAGGTAGCGAGTGGCTT ACAGGTAGGTGCGTGAAGCTTCTACAAGCA CAGTTGCCCCCTGGGAAGCA  Sequences marked with asterisk are C57BL/6-BALB/c junction sequences where C57BL/6 sequences are in parentheses. During cloning of the chimeric H-2Ea gene, exon 1 and the remainder of intron 1 of the C57BL/6 allele of H-2Ea was replaced with the equivalent 2616 bp region from the BALB/c allele of H-2Ea. This was done because exon 1 of the C57BL/6 allele of H-2Ea contains a deletion which renders the gene nonfunctional, while exon 1 of BALB/c allele of H-2Ea is functional. For a more detailed description, see U.S. Pat. No. 8,847,005, incorporated herein by reference.

The targeted BAC DNA described above was used to electroporate mouse ES cells comprising humanized MHC I (HLA-A2), as well as MHC II and H-2D deletion to create modified ES cells for generating mice that express chimeric MHC I and MHC II genes and lack functional endogenous mouse H-2E, H-2A, H-2K, and H-2D loci (see, e.g., step 5 in FIG. 3A). ES cells containing an insertion of human HLA sequences were identified by a quantitative PCR (TAQMAN™) assay, using primers and probes in Table 4.

TABLE 4 TAQMAN™ Primer and Probe Sequences for Detection of MHC I and MHC II Loci Humanization Name Forward Reverse (location) Primer Primer Probe Hyg cassette TGCGGCC TTGACCGA ACGAGC GATCTT TTCCTTG GGGTTC AGCC CGG GGCCC (SEQ ID (SEQ ID ATTC NO: 25) NO: 26) (SEQ ID NO: 27) 7092 hTUP1 CCCCACA CGTCCCAT TGGCAGC (Exon 2 of GCACGT TGAAGAA CTAAGA DRB1*1501) TTCCT ATGACACT GG (SEQ ID (SEQ ID (SEQ ID NO: 28) NO: 29) NO: 30) 7092 hTUP2 CCCCACA ACCCGCT AGCCTA (Exon 2 of GCACGT CCGTCCC AGAGGG DRB1*1501) TTCCT ATT AGTGTC (SEQ ID (SEQ ID (SEQ ID NO: 31) NO: 32) NO: 33) 7092 hTDP1 AGACCCT CGCTTGG TCGAAGT (Exon 3 of GGTGAT GTGCTCC GGAGA DRB1*1501) GCTGGAA ACTT GGTTTA (SEQ ID (SEQ ID (SEQ ID NO: 34) NO: 35) NO: 36) 7092 hTDP2 TGGAATG GCACGGT TGACTT (exon 3 of GAGTGAG CCCCTTC CCTAAAT DRB1M501) CAGCTTT TTAGTG TTCTC (SEQ ID (SEQ ID (SEQ ID NO: 37) NO: 38) NO: 39) hDRAIU CTGGCGG CATGATT CGATTT (exon 2 of CTTGAAG TCCAGGTT GCCAGCT DRA) AATTTGG GGCTTT TTGAGG (SEQ ID GTC CTCAAGG NO: 40) (SEQ ID (SEQ ID NO: 41) NO: 42) 1751jxn2¹ CCTCAC TTGTCCCA TGCATCT (loss-of- TTGGGA GTCACCG CGAGCAC allele assay, CCAACC TCCAT AGGCATT sequence CTA (SEQ ID TGG present in H- (SEQ ID NO: 44) (SEQ ID 2A and H-2E NO: 43) NO: 45) delete only) ¹A11 sequences except this one are used in the gain-of-allele assay.

The selection cassette may be removed by methods known by the skilled artisan. For example, ES cells bearing the chimeric human/mouse MHC class I locus may be transfected with a construct that expresses Cre in order to remove the “loxed” selection cassette introduced by the insertion of the targeting construct (see, e.g., step 6 in FIG. 3A). The selection cassette may optionally be removed by breeding to mice that express Cre recombinase. Optionally, the selection cassette is retained in the mice.

Targeted ES cells containing all of the modifications described herein (HLA-A2/H-2K, HLA-DR2/H-2E, H-2A-del, H-2D-del of FIG. 3A) were verified using a quantitative TAQMAN® assay described above using the primer/probe sets described herein for individual modifications. An additional primer/probe set was used to determine that during cassette-deletion step, no inverted clone was created due to lox sites present in opposing orientation.

Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007), supra). VELOCIMICE® (F0 mice fully derived from the donor ES cell) independently bearing a chimeric MHC class I and MHC II genes were identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the unique human gene sequences. A schematic representation of the genotype of MHC loci in the resulting mice is depicted in FIG. 3C (** represents H-2L gene which is not present in all mouse strains). Expression of both chimeric human/mouse MHC I and MHC II proteins is confirmed using antibodies specific for human HLA-DR2 and HLA-A2. Heterozygous mice are bred to homozygosity.

Example 1.4: Generation of Humanized β2 Microglobulin Mice

Generation of β2 microglobulin mice was described in U.S. Patent Application Publication No. 20130111617, incorporated herein by reference. Briefly, mouse β2 microglobulin (β2m) gene was humanized in a single step by construction of a unique targeting vector from human and mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al., supra).

Specifically, a targeting vector was generated by bacterial homologous recombination containing mouse β2m upstream and downstream homology arms from BAC clone 89C24 from the RPCI-23 library (Invitrogen). The mouse homology arms were engineered to flank a 2.8 kb human β2m DNA fragment extending from exon 2 to about 267 nucleotides downstream of non-coding exon 4 (FIG. 2C). A drug selection cassette (neomycin) flanked by recombinase recognition sites (e.g., loxP sites) was engineered into the targeting vector to allow for subsequent selection. The final targeting vector was linearized and electroporated into a F1H4 mouse ES cell line (Valenzuela et al., supra).

Targeted ES cell clones with drug cassette removed (by introduction of Cre recombinase) were introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al., supra). VELOCIMICE® (F0 mice fully derived from the donor ES cell) bearing the humanized β2m gene were identified by screening for loss of mouse allele and gain of human allele using a modification of allele assay (Valenzuela et al., supra). Heterozygous mice are bred to homozygosity. Expression of human β2 microglobulin was confirmed by flow cytometry using antibodies specific for human β2 microglobulin.

Example 2: Generation of Humanized T Cell Receptor Mice

Mice comprising a deletion of endogenous TCR (α or β) variable loci and replacement of endogenous V and J or V, D, and J segments are made using VELOCIGENE® genetic engineering technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela, D. M., et al. (2003), supra), wherein human sequences derived from BAC libraries using bacterial homologous recombination are used to make large targeting vectors (LTVECs) comprising genomic fragments of human TCR variable loci flanked by targeting arms to target the LTVECs to endogenous mouse TCR variable loci in mouse ES cells. Detailed description of the humanization of the TCR alpha and beta loci is described in U.S. Pat. No. 9,113,616, incorporated herein by reference. LTVECs re linearized and electroporated into a mouse ES cell line according to Valenzuela et al. ES cells are selected for hygromycin or neomycin resistance, and screened for loss of mouse allele or gain of human allele.

Targeted ES cell clones are introduced into 8-cell stage (or earlier) mouse embryos by the VELOCIMOUSE® method (Poueymirou, W. T. et al. (2007, supra). VELOCIMICE® (F0 mice fully derived from the donor ES cell) bearing humanized TCR loci are identified by screening for loss of endogenous TCR variable allele and gain of human allele using a modification of allele assay (Valenzuela et al., supra). F0 pups are genotyped and bred to homozygosity. Mice homozygous for humanized TCRα and/or TORβ variable loci are made as described herein.

Example 2.1: Humanization of TCR Alpha Locus

1.5 megabases of DNA at mouse TCRα locus corresponding to 110 V and 60 J mouse segments was replaced with 1 megabase of DNA corresponding to 54V and 61J segments of human TCRα using a progressive humanization strategy summarized in FIG. 4A and described in U.S. Pat. No. 9,113,616. Junctional nucleic acid sequences of various targeting vectors used for progressive humanization strategy of TCRα locus are summarized in Table 5, and included in the Sequence Listing.

TABLE 5 Junctional Nucleic Acid Sequences for Various TCRα Locus Targeting Vectors SEQ MAID ID NO. NO Description 1626 46 Junctional nucleic acid sequence between the 3′ end of mouse sequence upstream of the TCRα variable locus and the 5′ end of loxP-Ub-Hyg-loxP cassette. 47 Junctional nucleic acid sequence between the 3′ end of loxP-Ub-Hyg-loxP cassette and the 5′ end of human TCRVα40-TCRVα41-TCRJα1 insertion, including AsiSl site. 48 Junctional nucleic acid sequence between the 3′ end of human TCRVα40-TCRVα41-TCRJα1 insertion and the 5′ end of the mouse sequence downstream of the human TCRα variable locus, including Notl site. 1767 49 Junctional nucleic acid sequence between the 3′ end of mouse sequence upstream of the TCRα variable locus and the 5′ end of loxP-Ub-Neo-loxP cassette. 50 Junctional nucleic acid sequence between the 3′ end of loxP-Ub-Neo-loxP cassette and the 5′ end of human TCRVα35-TCRVα39 insertion, including AsiSl site. 1979 51 Junctional nucleic acid sequence between the 3′ end of mouse sequence upstream of the TCRα variable locus and the 5′ end of frt-Pgk-Hyg-frt cassette. 52 Junctional nucleic acid sequence between the 3′ end of frt-Pgk-Hyg-frt cassette and the 5′ end of human TCRVα22-TCRVα34 insertion, including AsiSl site. 1769 53 Junctional nucleic acid sequence between the 3′ end of mouse sequence upstream of the TCRα variable locus and the 5′ end of loxP-Ub-Neo-loxP cassette. 54 Junctional nucleic acid sequence between the 3′ end of loxP-Ub-Neo-loxP cassette and the 5′ end of human TCRVα13-2-TCRVα21 insertion, including AsiSl site. 1770 55 Junctional nucleic acid sequence between the 3′ end of mouse sequence upstream of the TCRα variable locus and the 5′ end of loxP-Ub-Hyg-loxP cassette. 56 Junctional nucleic acid sequence between the 3′ end of loxP-Ub-Hyg-loxP cassette and the 5′ end of human TCRVα6-TCRVα8-5 insertion, including AsiSl site. 1771 57 Junctional nucleic acid sequence between the 3′ end of mouse sequence upstream and the TCRα variable locus to the 5′ end of loxP-Ub-Neo-loxP cassette. 58 Junctional nucleic acid sequence between the 3′ end of loxP-Ub-Neo-loxP cassette and the 5′ end of human TCRVα1-1-TCRVα5 insertion, including AsiSl site. Human TCRα variable region segments are numbered as in IMGT database. At least 100 bp at each junction (about 50 bp from each end) are included in the Sequence Listing.

First, DNA from mouse BAC clone RP23-6A14 (Invitrogen) was modified by homologous recombination and used as a targeting vector to replace TCRAJ1-TCRAJ28 region of the endogenous mouse TCRα locus with a Ub-hygromycin cassette followed by a loxP site. DNA from mouse BAC clone RP23-117i19 (Invitrogen) was modified by homologous recombination and used as a targeting vector to replace ˜15 kb region surrounding (and including) TCRAV1 of the endogenous mouse TCRα and 8 locus with a PGK-neomycin cassette followed by a loxP site. ES cells bearing a double-targeted chromosome (i.e., a single endogenous mouse TCRα locus targeted with both of these targeting vectors) were confirmed by karyotyping and screening methods (e.g., TAQMAN™) known in the art. Modified ES cells were treated with CRE recombinase, thereby mediating the deletion of the region between the two loxP sites (i.e., the region consisting of the endogenous mouse TCRα locus from TCRAV1 to TCRAJ1) and leaving behind only a single loxP site, neomycin cassette and the mouse constant and enhancer regions. This strategy resulted in generation of a deleted mouse TCR α/δ locus (MAID 1540, FIG. 4A, second diagram).

The first human targeting vector for TCRα had 191,660 bp of human DNA from the CTD2216p1 and CTD2285m07 BAC clones (Invitrogen) that contained the first two consecutive human TCRα V gene segments (TRAV40 & 41) and 61 TCRαJ (50 functional) gene segments. This BAC was modified by homologous recombination to contain a Not1 site 403 bp downstream (3′) of the TCRαJ1 gene segment for ligation of a 3′ mouse homology arm and a 5′ AsiSI site for ligation of a 5′ mouse homology arm. Two different homology arms were used for ligation to this human fragment: the 3′ homology arm contained endogenous mouse TCRα sequences from the RP23-6A14 BAC clone and the 5′ homology arm contained endogenous TCRα sequence 5′ of mouse TCRαV from mouse BAC clone RP23-117i19. This mouse-human chimeric BAC was used as a targeting vector (MAID 1626) for making an initial insertion of human TCRα gene segments plus an upstream loxp-ub-hygromycin-loxp cassette at the mouse TCRα loci. The junctional nucleic acid sequences (SEQ ID NOs: 46-48) for the MAID 1626 targeting vector are described in Table 5.

Subsequently, a series of human targeting vectors were made that utilized the same mouse 5′ arm that contained endogenous TCRα sequence 5′ of mouse TCRαV from mouse BAC clone RP23-117i19 with alternating loxP-neomycin-loxP and loxP-hygromycin-loxP (or frt-hygromycin-frt for MAID 1979) selection cassettes. The specific constructs are described in U.S. Pat. No. 9,113,616, as well as depicted in FIG. 4A, with junctional sequences for each insertion included in Table 5 and the Sequence Listing. The final TCRα locus contained a 5′ loxp-ub-neomycin-loxP cassette plus a total of 54 human TCRαV (45 functional) and 61 human TCRαJ gene segment operably linked to mouse TCRα constant genes and enhancers. The junctional nucleic acid sequences (SEQ ID NOs: 57 and 58) for the MAID 1771 targeting vector are described in Table 5.

In any of progressive humanization steps, the selection cassettes are removed by deletion with Cre or Flp recombinase. In addition, human TCRδ locus may be reintroduced into the TCR alpha sequence.

Example 2.2: Humanization of TCRβ Variable Locus

The organization of the TRB locus is more complex. In both mouse and human, most V gene segments are located between two large clusters of Trypsinogen (Try) genes, and two clusters of D, J, C genes are located between the second Try cluster and the 3′ enhancer. All mouse Trbv, Trbd, and Trbj genes, except for the most distal Trbv1 gene, were humanized in nine steps (FIG. 4B). The mouse Trbv1 gene was not humanized because it is used at a very low frequency and has no human ortholog. To humanize the Trbd and Trbj genes, two different animals were created. In one, the mouse TCRBDJ1 and TCRBDJ2 regions were fully humanized, e.g., comprised contiguous human TCRBDJ1 and TCRBDJ2 sequences, e.g., comprised human TCRBDJ1 and TCRBDJ2 non-coding sequences (2 functional human TCRBD segments and 14 (13 functional) human TCRBJ segments). In the other, the TCRBDJ1 and TCRBDJ2 regions were modified such that the mouse coding sequences were precisely replaced with the equivalent human coding regions, with all of the non-coding sequences remaining mouse. Finally, in both animals, the inverted mouse Trbv31 gene downstream of the 3′ enhancer was replaced with the equivalent human TRBV30 gene.

More specifically, 0.6 megabases of DNA at mouse TCRβ locus corresponding to 33 V, 2 D, and 14 J mouse segments were replaced with 0.6 megabases of DNA corresponding to 67 V, 2D, and 14 J segments of human TCRβ using a progressive humanization strategy summarized in FIG. 4B and described in detail in U.S. Pat. No. 9,113,616. Junctional nucleic acid sequences of various targeting vectors used for progressive humanization strategy of TCRβ locus are summarized in Table 6, and included in the Sequence Listing.

TABLE 6 Junctional Nucleic Acid Sequences for Various TCRβ Locus Targeting Vectors SEQ MAID ID NO. NO Description 1625 59 Junctional nucleic acid sequence between the 3′ end of mouse sequence upstream of the TCRβ variable locus (nearby the upstream mouse trypsinogen genes) and the 5′ end of frt-Ub-Neo-frt cassette. 60 Junctional nucleic acid sequence between the 3′ end of frt-Ub-Neo-frt cassette and the 5′ end of human TCRVβ18-TCRVβ29-1 insertion. 61 Junctional nucleic acid sequence between the 3′ end of human TCRVβ18-TCRVβ29-1 insertion and the 5′ end of the mouse sequence downstream of the mouse TCRVβ segments (nearby downstream mouse trypsinogen genes). 1715 62 Junctional nucleic acid sequence between 3′ of the downstream mouse trypsinogen genes and the 5′ end of human TCRDβ1-TCRJβ1-1-TCRJβ1-6 insertion, including Iceul site. 63 Junctional nucleic acid sequence between the 3′ end of human TCRDβ1-TCRJβ1-1-TCRJβ1-6 insertion and the 5′ end of loxP-Ub-Hyg-loxP cassette. 64 Junctional nucleic acid sequence between the 3′ end of loxP-Ub-Hyg-loxP cassette and the 5′ end of mouse sequence nearby the mouse Cβ1 gene. 65 Junctional nucleic acid sequence between the 3′ end of the mouse sequence nearby the mouse Cβ1 gene and the 5′ end of human TCRDβ2-TCRJβ2-1-TCRJβ2-7 insertion, including Notl site. 66 Junctional nucleic acid sequence between the 3′ end of human TCRDβ2-TCRJβ2-1-TCRJβ2-7 insertion and the 5′ end of the mouse sequence downstream of the TCRβ variable locus (nearby the Cβ2 mouse sequence). 1791 67 Junctional nucleic acid sequence between the 3′ end of mouse sequence upstream of the TCRβ variable locus (nearby the upstream mouse trypsinogen genes) and the 5′ end of frt-Ub-Hyg-frt cassette. 68 Junctional nucleic acid sequence between the 3′ end of frt-Ub-Hyg-frt cassette and the 5′ end of human TCRVβ6-5-TCRVβ17 insertion. 1792 69 Junctional nucleic acid sequence between the 3′ end of mouse sequence upstream of the TCRβ variable locus (nearby the upstream mouse trypsinogen genes) and the 5′ end of frt-Ub-Neo-frt cassette. 70 Junctional nucleic acid sequence between the 3′ end of frt-Ub-Hyg-frt cassette and the 5′ end of human TCRVβ1-TCRVβ12-2 insertion. 6192 71 Junctional nucleic acid sequence between the 3′ end of mouse sequence nearby the mouse Cβ2 gene and the 5′ end of the human TCRBV30 exon 2 sequence. 72 Junctional nucleic acid sequence between the 3′ end human TCRBV30 exon 1 sequence and the 5′ end of mouse sequence downstream of TCRβ locus. Human TCRβ variable region segments are numbered as in IMGT database. At least 100 bp at each junction (about 50 bp from each end) are included in the Sequence Listing.

Specifically, DNA from mouse BAC clone RP23-153p19 (Invitrogen) was modified by homologous recombination and used as a targeting vector to replace 17 kb region (including TCRBV30) just upstream of the 3′ trypsinogen (TRY) gene cluster in the endogenous mouse TCRβ locus with a PGK-neo cassette followed by a loxP site. DNA from mouse BAC clone RP23-461 h15 (Invitrogen) was modified by homologous recombination and used as a targeting vector to replace 8355 bp region (including TCRBV2 and TCRBV3) downstream of 5′ trypsinogen (TRY) gene cluster in the endogenous mouse TCRβ locus with a Ub-hygromycin cassette followed by a loxP site. ES cells bearing a double-targeted chromosome (i.e., a single endogenous mouse TCRβ locus targeted with both targeting vectors) were confirmed by karyotyping and screening methods (e.g., TAQMAN™) known in the art. Modified ES cells were treated with CRE recombinase, mediating the deletion of the region between the 5′ and 3′ loxP sites (consisting of the endogenous mouse TCRβ locus from TCRBV2 to TCRBV30) and leaving behind only a single loxP site, hygromycin cassette and the mouse TCRBDs, TCRBJs, constant, and enhancer sequences. One mouse TCRVβ was left upstream of the 5′ cluster of trypsinogen genes, and one mouse TCRVβ was left downstream of the mouse Eβ, as noted in FIG. 4B.

The first human targeting vector for TCRβ had 125,781 bp of human DNA from the CTD2559j2 BAC clone (Invitrogen) that contained the first 14 consecutive human TCRβV gene segments (TRBV18-TRBV29-1); the junctional nucleic acid sequences (SEQ ID NOs: 59-61) for the MAID 1625 targeting vector are described in Table 6.

In order to replace mouse TCRβ D and J segments with human TCRβ D and J segments, DNA from mouse BAC clone RP23-302p18 (Invitrogen) and from human BAC clone RP11-701D14 (Invitrogen) was modified by homologous recombination and used as a targeting vector (MAID 1715) into the ES cells that contained the TCRβV locus described above (i.e., MAID 1625). This modification replaced a contiguous ˜18540 bp region (from 100 bp downstream of the polyA of the 3′ trypsinogen genes to 100 bp downstream from the J segments in the D2 cluster which included mouse TCRBD1-J1, mouse constant 1, and mouse TCRBD2-J2) in the endogenous mouse TCRβ locus with ˜25425 bp of sequence containing human TCRBD1-J1, loxP Ub-hygromycin-loxP cassette, mouse constant 1, human TCRBD2-J2. ES cells bearing a double-targeted chromosome (i.e., a single endogenous mouse TCRβ locus targeted with both targeting vectors) were confirmed by karyotyping and screening methods (e.g., TAQMAN™) known in the art. Modified ES cells were treated with CRE recombinase thereby mediating the deletion the hygromycin cassette leaving behind only a single loxP site downstream from human J segments in D1J cluster. The junctional nucleic acid sequences (SEQ ID NOs: 62-66) for the MAID 1715 targeting vector are described in Table 6.

Subsequently, a series of human targeting vectors were made that utilized the same mouse 5′ arm that contained endogenous TCRβ sequence surrounding the upstream mouse trypsinogen genes from mouse BAC clone RP23-461 h15 with alternating selection cassette. The specific constructs are described in U.S. Pat. No. 9,113,616, as well as depicted in FIG. 4B, with junctional sequences for each insertion included in Table 6 and the Sequence Listing.

Finally, a human TCRβ locus containing a total 66 human TCRβV (47 functional) and the human TCRβ D and J segments (MAID 1792) operably linked to mouse TCRβ constant genes and enhancers was generated. The junctional nucleic acid sequences (SEQ ID NOs: 69 and 70) for the MAID 1792 targeting vector are described in Table 6.

Mouse TCRBV31 is located ˜9.4 kb 3′ of TCRBC2 (second TCRB constant region sequence) and is in the opposite orientation to the other TCRBV segments. The equivalent human V segment is TCRBV30, which is located in a similar position in the human TCRB locus. To humanize TCRBV31, the mouse BAC clone containing mouse TCRBV31, was modified by bacterial homologous recombination to make LTVEC MAID 6192. The entire coding region, beginning at the start codon in exon 1, the intron, the 3′ UTR, and the recombination signal sequences (RSS) of TCRBV31 were replaced with the homologous human TCRBV30 sequences. FIG. 4B depicts the selection cassette located in the intron between exon 1 and exon 2 of the hTCRBV30 gene.

The junctional nucleic acid sequences (SEQ ID NOs: 71 and 72) for the MAID 6192 targeting vector are described in Table 6. MAID 6192 DNA is electroporated into MAID1792 ES cells, and cells are screened for loss of mouse TCRB31 allele and gain of human TCRB30 allele.

Replacement of a contiguous ˜18540 bp region (from 100 bp downstream of the polyA of the 3′ trypsinogen genes to 100 bp downstream from the J segments in the D2 cluster) in the endogenous mouse TCRβ locus with a ˜25425 bp of sequence containing a contiguous human sequence comprising human TCRBD1-J1 gene segments, loxP Ub-hygromycin-loxP cassette, mouse constant 1, and a contiguous human sequence comprising human TCRBD2-J2 gene segments as described herein resulted in mice comprising fully human TCRBDJ1 and TCRBJ2 clusters. Breeding mice with fully human TCRBDJ1 and TCRBJ2 clusters with mice modified to express other human or humanized components, e.g., MHC I, MHC II α and β, TCRα, CD4, CD8α and β, and β2M in any combination, created mice that expressed relatively normal B and T cell markers and populations, have normal T cell function intact memory T cell responses, are capable of processing LCMV expressed protein (see, e.g., Examples 6-7). The mice comprising fully human TCRBDJ1 and TCRBDJ2 clusters demonstrated significant gene usage from the TCRBDJ1 cluster but not the TCRDJ2 cluster (data not shown). To address this, mice comprising TCRB non-coding sequences at the TCRDJ1 and/or TCRDJ2 clusters may be made.

Specifically, mouse Trbd1, Trbj1-1 to 1-6 gene segment coding regions (coordinates chr6: 41,533,201-41,535,606, +strand; GRCm38 assembly, NCBI Accession Number NC_000072.6) were replaced with human TRBD1, TRBJ1-1 to 1-6 gene segment coding regions (coordinates chr7: 142,786,213-142,789,040, +strand, GRCh38 assembly, NCBI Accession Number NC_000007.14); and mouse Trbd2, Trbj2-1 to 2-7 gene segment coding regions (coordinates chr6: 41,542,163-41,543,856, GRCm38 assembly, NCBI Accession Number NC_000072.6) were replaced with human TRBD2, and TRBJ2-1 to 2-7 gene segment coding regions (coordinates chr7: 142,795,686-142,797,502+strand, GRCh38 assembly, NCBI Accession Number NC_000007.14). Specific replaced coding sequences and their coordinates are indicated in Table 7 below.

TABLE 7 Replacement of mouse Trb D and J gene segments with human TRB D and J gene segments Modified mouse Mouse genome coordinates Human gene Human genome coordinates gene name (chr6, GRCh38) name (chr7, GRCh38) Trbd1 6: 41,533,201-41,533,212 TRBD1 7: 142,786,213-142,786,224 Trbj1-1 6: 41,533,864-41,533,911 TRBJ1-1 7: 142,786,880-142,786,927 Trbj1-2 6: 41,534,001-41,534,048 TRBJ1-2 7: 142,787,017-142,787,064 Trbj1-3 6: 41,534,323-41,534,376 TRBJ1-3 7: 142,787,630-142,787,679 Trbj1-4 6: 41,534,811-41,534,861 TRBJ1-4 7: 142,788,225-142,788,275 Trbj1-5 6: 41,535,084-41,535,133 TRBJ1-5 7: 142,788,498-142,788,547 Trbj1-6 6: 41,535,554-41,535,606 TRBJ1-6 7: 142,788,988-142,789,040 Trbd2 6: 41,542,163-41,542,176 TRBD2 7: 142,795,686-142,795,740 Trbj2-1 6: 41,542,754-41,542,803 TRBJ2-1 7: 142,796,365-142,796,414 Trbj2-2 6: 41,542,957-41,543,007 TRBJ2-2 7: 142,796,560-142,796,610 Trbj2-3 6: 41,543,223-41,543,271 TRBJ2-3 7: 142,796,847-142,796,895 Trbj2-4 6: 41,543,362-41,543,410 TRBJ2-4 7: 142,796,998-142,797,047 Trbj2-5 6: 41,543,453-41,543,501 TRBJ2-5 7: 142,797,119-142,797,166 Trbj2-6 6: 41,543,598-41,543,645 TRBJ2-6 7: 142,797,239-142,797,291 Trbj2-7 6: 41,543,810-41,543,856 TRBJ2-7 7: 142,797,456-142,797,502

Two constructs, one comprising chimeric TRBDJ1 region, and one comprising chimeric TRBDJ2 region, were synthesized de novo by Blue Heron Biotechnology. In these constructs, only the specific D and J segments listed above in Table 7 are human, and the non-coding sequences between them, including RSSs and other intergenic sequences, are mouse. Blue Heron constructs were synthesized with 5′ and 3′ mouse homology arms, which also served as PCR primers for amplifying the chimeric region and for various subsequent steps of bacterial homologous recombination (BHR) (see Table 8; “homology boxes”). A selection cassette was added to each construct to aid in clone selection.

TABLE 8 Mouse Homology boxes (BHR) for Introducing Chimeric TRBDJ Sequences 5′ box 3′ box coordi- coordi- 5′ nates 3′ nates Homology homology (GRCm38 homology (GRCm38 boxes box assembly) box assembly) Trbdj1 AACTC chr6: ACTGA chr6: CCCCC 41533101- AGACA 41535788- CAGAG 41533201 GTAGC 41535887 GAGCA CCAAA GCTTA GAATA TCTGG GTAAA TGGTT AAGAA TCTTC GAGCA CAGCC AGAAG CTCAA ACTCA GGGGT AAGCC AGACC CTGAA TATGG GAAAG GAGGG GCATT TCCTT CTGTG TTTTG TCCAG TATAA AACAC AGCTG GAAAA TAACA CAAAC TTGTG CCAGA GAAAC ATTAT CTTGC TTGCC GCTCG GACTA TTCGC CCTTG (SEQ ID (SEQ ID NO: 92) NO: 93) Trbdj2 AGACA chr6: AACCT chr6: AACCT 41542063- ACCTT 41543957- CTCTG 41542163 GTCCT 41544056 CCACC GGCTT TGGTC GCGAG TCCCT AGAGC GCCCC GAACA TGCCC ACGTT AGGCT TGGCA CTGGG CCCAA GTAGG TCCCT CACCT TTTCA GTGGG TCCCG GAAGA CTCCC AACTT CCAGA TTTTG AAGGG TATCA TGAAG CGATG TTGAG TAACA AGCTG TTGTG TCTCC GAAAC ATTAT CTTGC TTGCC GCTCG GACTA TTCGC CCTTG (SEQ ID (SEQ ID NO: 94) NO: 95)

In detail, in a first BHR step, a spectinomycin resistance cassette was inserted in place of mouse Trbdj1 gene segment region in mouse BAC RP23-302p18, using the Trbdj1 homology boxes from Table 8. The resulting construct contained, from 5′ to 3′: a chloramphenicol-resistance cassette, the 5′ arm, a spectinomycin-resistance cassette, and the 3′ arm. This construct was used as the backbone into which a nucleotide sequence comprising (1) ˜2.7 kb region of chimeric TRBD1, TRBJ1-1 to 1-6 from the first Blue Heron construct and (2) a hygromycin-resistance cassette, in place of spectinomycin-resistance cassette, is inserted in a second BHR step. A third BHR step included insertion of another spectinomycin-resistance cassette in place of a mouse Trbd2 gene segment region in the construct comprising the chimeric Trbdj1 gene segments described above, using the Trbdj2 homology boxes from Table 8. Finally, a nucleotide sequence comprising (1) ˜2 kb region of chimeric TRBD2, and TRBJ2-1 to 2-7 from the second Blue Heron construct and (2) a neomycin resistance cassette was inserted via BHR, in place of the second spectinomycin-resistance cassette. Selection cassettes may be removed at different steps using various methods known in the art (e.g., introduction of Cre recombinase, restriction digestion, etc.)

The final targeting vector contained, from 5′ to 3′: chloramphenicol-resistance cassette, ˜2.7 kb of chimeric human sequence of TRBDJ1, a loxp-Ub-hyg-lox cassette, mouse Trbc1 constant gene (GRCm38 coordinates chr6: 41,538,218-41,539,347), ˜2 kb of chimeric human genomic sequence of TRBDJ2, and mouse Trbc2 constant gene (GRCm38 coordinates chr6: 41,546,730-41,548,352). Final targeting vector clone was selected based on chloramphenicol/hygromycin resistance.

This final targeting vector was electroporated into mouse embryonic stem (ES) cells comprising the locus depicted in FIG. 4B, bottom schematic (MAID 6192), after recombinase mediated deletion of the hygromycin and neomycin resistance cassettes in that locus. Targeted homologous recombination resulted in

-   -   (1) deletion of ˜22.8 kb of a sequence comprising         -   the human TRBDJ1 region (GRCm38 coordinates chr7:             142,775,154-142,789,107),         -   a mouse constant Trbc1 gene, and         -   ˜4.6 kb of the human TRBDJ2 region (GRCm38 coordinates chr7:             142,793,139-142,797,737), and     -   (2) insertion of ˜19.1 kb of a sequence comprising         -   ˜a TRBDJ1 cluster that comprises 2.7 kb of a sequence             comprising human TRBD1 and human TRBJ1-1 to TRBJ1-6 gene             segments and mouse TCR non-coding sequences, where the human             TRBD1 and human TRBJ1-1 to TRBJ1-6 gene segments flank the             same mouse TCR non-coding sequences as are normally flanked             by mouse Trbd1 and mouse Trbj1-1 to Trbj1-6 gene segments,         -   a mouse constant Trbc1 gene, and         -   ˜a TRBDJ2 cluster that comprises 2 kb of a sequence             comprising human TRBD2 and human TRBJ2-1 to TRBJ2-7 gene             segments and mouse TCR non-coding sequences, where the human             TRBD2 and human TRBJ2-1 to TRBJ2-7 gene segments flank the             same mouse TCR non-coding sequences as are normally flanked             by the mouse Trbd2 and mouse Trbj2-1 to Trbj2-7 gene             segments (FIG. 4C).             Successful integration was confirmed by a modification of             allele (MOA) assay as described, e.g., in Valenzuela et al,             supra, using primers and probes for inserted or deleted             sequences.

Positively targeted ES cells were used as donor ES cells and microinjected into a pre-morula (8-cell) stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. Nos. 7,576,259, 7,659,442, 7,294,754, and US 2008-0078000 A1, all of which are incorporated herein by reference). The mouse embryo comprising the donor ES cells was incubated in vitro and then implanted into a surrogate mother to produce an F0 mouse fully derived from the donor ES cells. Mice bearing a humanized TCRB locus (with TRBDJ1 and 2 regions comprising murine TCRB non-coding sequences) were identified by genotyping using the MOA assay. Mice heterozygous for the humanized TCRB locus (with TRBDJ1 and 2 regions comprising murine TCRB non-coding sequences) were bred to homozygosity.

The final humanized TCRβ locus mouse comprising murine TCR non-coding sequences in the TRBDJ1 and 2 regions comprised human TCRB V region (67 TRBV gene segments), two TCRBDJ regions (total 2 functional TRBDs and β functional TRBJ segments, with mouse TCRB non-coding sequences in each of the TRBDJ1 and 2 regions), mouse constant Trbc1 and Trbc2 genes, and mouse enhancers.

Similar engineering strategy to the one described above is used to optionally delete the remaining 5′ mouse TCRβ V segment.

In any of the above steps, the selection cassettes are removed by deletion with Cre or Flp recombinase, and other methods known in the art.

Mice homozygous for humanized TCRα variable locus are bred with mice homozygous for humanized TCRβ variable locus to form progeny comprising humanized TCRα and TCRβ variable loci. Progeny are bred to homozygosity with respect to humanized TCRα and humanized TCRβ loci.

Mice comprising humanized TCRα and TCRβ variable loci are confirmed to undergo normal T cell development and comprise T cell receptors that express variable domains derived from a variety of variable gene segments.

Example 3: Humanization of T Cell Co-Receptor Loci

Humanization of CD4 and CD8 loci (both CD8alpha and CD8 beta loci) is described in detail in U.S. Patent Application Publication No. 20140245466, incorporated herein in its entirety by reference.

Example 3.1: Humanization of CD4 Locus

Specifically, mouse CD4 locus was humanized in a single step by construction of a unique targeting vector from human and mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003), supra). To generate the targeting vector, a series of bacterial homologous recombinations (BHRs) using Bacterial Artificial Chromosome (BAC) DNA, as well as other engineering steps, were carried out as described in detail in U.S. Patent Application Publication No. 20140245466.

The human CD4 Targeting Vector was linearized with Notl and electroporated into F1H4 mouse ES cells. Targeted ES cells bearing a humanized CD4 locus were identified by genotyping using a modification of allele assay (Valenzuela et al.) that detected the presence of the neomycin cassette and the human CD4 gene, as well as one copy of the mouse CD4 gene.

The final humanized CD4 locus derived from successful incorporation of humanized CD4 targeting vector into ES cells is depicted in FIG. 5A. The sequence across the human intron 3—lox-neo cassette junction (5′ end of the cassette) is set forth in SEQ ID NO:75, and the sequence across lox-neo cassette—human intron 3 junction (3′ end of the cassette) is set forth in SEQ ID NO:76; both sequences are also listed in Table 9. The complete nucleic acid sequence of the humanized CD4 piece, including the pgk-neo cassette depicted in FIG. 5A is set forth in SEQ ID NO:77. The pgk-neo cassette is spans residues 307-2176 of SEQ ID NO:77, the two lox sites are located at residues 267-300 and 2182-2215, while the human sequence spans residues 1-234 and 2222-18263. The amino acid sequence of complete humanized CD4 protein is set forth in SEQ ID NO:78, with human sequence spanning amino acids 27-319 (set forth in SEQ ID NO:79).

TABLE 9 Junction Sequences of the Chimeric CD4 Targeting Vector SEQ ID Junction Sequence NO 5′ mouse/ AGGGGAAACCCGCAAAGGATGGG 73 human ACATAGGGAGACAGCTGTTAACA junction TCTGAAACATGACCTTCTTTTCT GTGCAGCACAACTCCTAGCTGTC ACTCAAGGG(AAGAAAGTGGTGC TGGGCAAAAAAGGGGATACAGTG GAACTGACCTGTACAGCTTCCCA GAAGAAGAGCATACAATTCCACT GGAAAAACTCCAACCAGAT) 3′ human/ (CTGGTCACCTGGATGAAGTGAG 74 mouse GGAGGGCCCTCTGGGTTTGGGGC junction TGGTTTTGAACTGAGACATCCAT GAGCCAGCCTGGGGCTGGCTTCA CTGAAGATC)ATCTATGTCGGGT GCGGAGAAAGAGGTAATGAAATG GCACATGCTATGTACAAACTCTA TTGCTGAGCAGCACCCAGTCCTG AGCTGGCTCTGAATTGAGGGTGA AATTCACACATTCTCCCCCAACA TCTATAATCTGG Human/5′ (TATGGAGTGAAAGCCTTTGGTG 75 lox site TCTGAGATCTGGTCTTAGTTAAA CTCTGGGATC)GGCGCGCCGAAT TCCTGCAGCCCGGGCTCGAGATA ACTTCGTATAATGTATGCTATAC GAAGTTATATGCATCCGGGTAGG GGAGGCGCTTTTCCC 3′ lox site/ AGTATTGTTTTGCCAAGTTCTAA human TTCCATCAGACCTCGACCTGCAG 76 CCCTAGATAACTTCGTATAATGT ATGCTATACGAAGTTATCCTAGG (CCAGAGGGCTTGGGTTGACAGA AACTCAGTGGCATTCTTATCCAG AGTTTCTCTACACC) Human sequences are in parenthesis and sequence containing restriction enzyme site (PI-Sce I) is bolded. Selection cassette sequences are italicized.

Floxed neomycin resistance cassette is removed by electroporation of plasmid expressing Cre recombinase into ES cells containing humanized CD4 locus.

Targeted ES cells bearing a humanized CD4 locus without resistance marker are identified by genotyping that detected absence of the neomycin cassette, the presence of one copy of the human CD4 gene and one copy of the mouse CD4 gene.

Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007, supra). VELOCIMICE® (F0 mice fully derived from the donor ES cell) independently bearing a chimeric CD4 gene were identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the unique human CD4 gene sequences. Expression of humanized CD4 proteins on the surface of T cells was detected using anti-human CD4 antibodies. Mice heterozygous for humanized CD4 protein described herein were bred to homozygosity.

Example 3.2: Humanization of CD8 Loci

CD8α and CD8β genes are colocalized in the genome, e.g., on mouse chromosome 6, they are located about 37 kb away from each other. Due to close linkage, sequential targeting, by first introducing one gene, e.g., CD8β, followed by introduction of the second gene, e.g., CD8α, is performed. Specific detailed steps of humanization are described in U.S. Patent Application Publication No. 20140245466, incorporated herein by reference.

Briefly, mouse CD8β locus was humanized in a single step by construction of a unique targeting vector from mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology. DNA from BAC RP23-431M6 was modified by BHR to generate a large targeting vector (LTVEC), MAID 1737, to contain a replacement of mouse exons 2-3 encoding the CD8 ecto domain (from the 5′ junction in intron 1 to the 3′ junction in intron 3), with homologous human sequences (FIG. 5B). A loxp-Ub-Hyg cassette was inserted at the 3′ junction in intron 3. The nucleotide sequence at various junctions of the resulting vector are listed in Table 10 and set forth in Sequence Listing. The complete amino acid sequence of humanized CD8β protein is set forth in SEQ ID NO:83; with human sequences spanning amino acids 15-165 (set forth in SEQ ID NO:84).

TABLE 10 Junction Sequences of the Chimeric CD8p Targeting Vector SEQ ID Junction Sequence NO Mouse/human in TGTTTGCCTGTGACATGAAC 80 intron 1 TCATTGTGACACAAACCACT GTGCTAGGGGGGATCCACTA GTAACGGCCGCCAGTGTGCT GGAATTCGCCC(TCGCAAGG GCCAGGCATATAAGTACACA ATAAACAAATGGCAGCTCTC TCC) Human/5′ of lox (CCCCTCCTTCCTTCCCCAG 81 site in intron 3 GCACTTTCCAAGTGTCAACT CTAGAGCCTAT)CGCGGCCG CACCGGT ATAACTTCGTATA ATGTATGCTATACGAAGTTA T 3′ of lox ATAACTTCGTATAATGTATG 82 site/mouse in CTATACGAAGTTAT GTCGAC intron 3 GTAGCCTATTTCTCTAGATC CAAAATGATGACAACAAAAG GTACCTTGTG Human sequences are in parenthesis, lox sites are italicized, and restriction enzyme sites, multiple cloning sites, and vector-derived sequences are bolded.

Targeting vector was electroporated into F1H4 mouse ES cells. Targeted ES cells bearing a humanized CD8β locus were identified by genotyping using a modification of allele assay (Valenzuela et al.) that detected the presence of the human CD8β gene.

Mouse CD8α locus was also humanized in a single step by construction of a unique targeting vector from mouse bacterial artificial chromosome (BAC) DNA using VELOCIGENE® technology. DNA from BAC RP23-431M6 was modified by BHR to generate a large targeting vector (LTVEC), MAID 1738, to contain a replacement of mouse exons 1-2 encoding the CD8a ecto domain (from the 5′ junction at Ala codon 27 in mouse exon 1 to the 3′ junction in mouse intron 2), with the homologous human sequences (from the 5′ junction in human exon 2 to the 3′ junction in intron 3 (FIG. 5A)). This retains the mouse leader sequence at the beginning of exon 1. A lox2372-Ub-Neo cassette was inserted at the 3′ junction of human/mouse sequences. The nucleotide sequences at various junctions of the resulting vector are listed in Table 11 and set forth in Sequence Listing. The complete amino acids sequence of humanized CD8α polypeptide is set forth in SEQ ID NO:88, with human sequence spanning amino acids 28-179 (set forth in SEQ ID NO:89).

TABLE 11 Junction Sequences of the Chimeric CD8α Targeting Vector SEQ ID Junction Sequence NO Mouse/human TGAACCTGCTGCTGCTGGG 85 at exon 1 TGAGTCGATTATCCTGGGG (mouse) and AGTGGAGAAGCT(AGGCCG exon 2 AGCCAGTTCCGGGTGTCGC (human) CGCTGGATCGGACCTGGAA CCTGGG) Human/5′ of (ATGCCAGGGACAGCCCTG 86 lox 2372 site ATACTGTAGGTAGAGTCAA GGGCTGTCCAAGT)ACCGG T ATAACTTCGTATAAGGTA TCCTATACGAAGTTAT 3′ of lox 2372 ATAACTTCGTATAAGGTAT 87 site/mouse CCTATACGAAGTTAT CTCG ACCTGATCTTGGAGGGAGA CCTGGACCGGGAGACGTGC TGGGGGCAGGGTT Human sequences are in parenthesis, lox sites are italicized, and restriction enzyme sites, multiple cloning sites, and vector-derived sequences are bolded.

Humanized CD8α targeting vector described above was electroporated into mouse ES cells that contained a humanized CD8b locus to create modified ES cells that comprise humanized CD8b and CD8a loci (FIG. 5B). Targeted ES cells bearing a humanized CD8a and CD8b loci were identified by genotyping using a modification of allele assay (Valenzuela et al.).

Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al, supra). VELOCIMICE® (F0 mice fully derived from the donor ES cell) bearing a chimeric CD8b gene and a chimeric CD8a gene were identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the unique human CD8b and CD8a gene sequences.

The selection cassettes in CD8α and CD8β loci may be removed by methods known by the skilled artisan. Mice heterozygous for humanized CD8α and CD8β loci as described herein are bred to homozygosity. Expression of humanized CD8α and CD8β on the surface of T cells is detected using anti-human CD8 antibodies.

Example 4: Generation of Mice Comprising Humanized Cellular Immune System Components

In order to generate mice comprising humanized cellular immune system components, mice homozygous for humanization of various components, e.g., MHC I, MHC II α and β, TCRα and β (e.g., mice homozygous for humanized TCRβ loci comprising either fully human TCRBDJ1 and TCRBDJ2 clusters or e.g., mice homozygous for humanized TCRβ loci comprising humanized TCRBDJ1 and TCRBDJ2 clusters with murine TCRB noncoding sequences and human coding sequences), CD4, CD8α and β, and β2M may be bred together in any combination to create mice that have different components of the T cell immune response humanized. For example, a mouse comprising a humanized MHC I may be bred with a mouse comprising a humanized β2M to generate a mouse expressing humanized MHC I/β2M. Mice homozygous for humanization of various components, e.g., MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M are bred together using methods known in the art to obtain a mouse comprising all nine humanizations (“TM I/II B C4/8” mice, which may also be referred to herein as “VelociT” mice). Mice are bred to homozygosity using methods known in the art. Alternatively, targeting vectors comprising each humanized gene can be introduced via sequential targeting into the same ES cell to obtain an ES cell comprising all nine humanizations, and the resultant ES cell is introduced into 8-cell stage mouse embryo by the VELOCIMOUSE® method, described in Examples 1-3 above. TM I/II B C4/8 mice may comprise fully human TCRBDJ1 and TCRBDJ2 clusters (see, e.g., Examples 5-7 and associated figures) or may comprise humanized TCRBDJ1 and TCRBDJ2 clusters comprising murine TCRBDJ1 and TCRBDJ2 noncoding sequences and human TCRBDJ1 and TCRBDJ2 coding sequences (see, e.g., Example 8 and associated figures).

Example 5: Characterization of Mice Comprising Humanized Cellular Immune System Components

Mice homozygous for humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β and for humanized β2M were characterized. Specifically, spleen and thymi, from mice were harvested and single cell suspensions were obtained. Suspensions were centrifuged at 1200 rpm for 5 min at 4° C. to pellet cells, and cells from each tissue were lysed with 4 mL of ACK lysing buffer (GIBCO) to lyse red blood cells. Cells were filtered through cell strainer, centrifuged to pellet, resuspended in media and counted.

Cell surface expression of CD19, CD3, CD4 and CD8α depicted in FIGS. 6A-C and FIGS. 9A-C was analyzed by FACS using fluorochrome-conjugated antibodies: anti-mouse CD3 (17A2, BD), anti-mouse CD19 (1D3, BD), anti-mouse F4/80 (BM8, Biolegend), anti-mouse CD8α (53-6.7, BD), anti-mouse CD4 (RM4-5, eBioscience), anti-human CD8α (SK1, BD), and anti-human CD4 (RPA-T4, BD). Cell surface expression of mouse H2db, human HLA molecules (HLA-A2, B2m, and HLA-DR) and mouse MHC I^(A)I^(E) molecules in FIGS. 7A-F and 10A-F was analyzed by FACS using fluorochrome-conjugated antibodies: anti-mouse CD19 (6D5, Biolegend), anti-mouse F4/80 (BM8, Biolegend), anti-mouse H2db (KH95, Biolegend), anti-human HLA-A2 (BB7.2, BD), anti-human HLA-DR (G46-6, BD), anti-human B2-mibroglobulin (2M2, Biolegend) and anti-mouse I^(A)I^(E) (M5/114.15.2, eBioscience). Cell surface expression of mouse and human CD4 and CD8 in FIG. 7G and FIG. 10G was analyzed by FACS using fluorochrome-conjugated antibodies: anti-mouse CD3 (17A2, Biolegend), anti-mouse CD4 (GK1.5, eBiosciences), anti-mouse CD8α (53-6.7, BD 2), anti-mouse CD8β (H35-17.2, eBioscience), anti-human CD4 (OKT4, eBioscience), anti-human CD8α (RPA-T8, BD 6), anti-human CD8β (2ST8.5H7, BD). Cell surface expression of FoxP3 and CD25 shown in FIG. 8 or FIG. 11 was analyzed by FACS anti-FoxP3 (FJK-16s, eBioscience) and anti-CD25 (PC61, Biolegend) Cell surface expression of CD44 and CD62L shown in FIGS. 9D-9E was analysed using anti-CD44 (IM7, BD) and anti-CD62L (MEL-14, Biolegend).

All flow cytometry was performed using BD Fortessa. Data was analyzed using FlowJo.

Expression in thymus is depicted in FIGS. 6A-C, 7A-G and 8. The absolute numbers of thymocytes and CD3⁺ cells, and the overall development of thymic T cells, were comparable in control mice and humanized TM I/II B C4/8 mice (data not shown). FIG. 6A shows that the proportion of B cells and T cells in the thymi of mice having a humanized cellular immune system (TM I/II B C4/8) is similar to the proportion found in control mice. The frequency and number of F4/80 cells in the thymi of TM I/II B C4/8 mice was compared to control mice (FIG. 6B and data not shown). Also, humanized CD4 and CD8 are expressed on thymic cells of a mouse humanized for all nine cellular immunity genes (TM I/II B C4/8), similar to the expression of mouse CD4 and CD8 in non-humanized control mice (FIG. 6C). Humanized β2M is expressed on the surface of B cells and macrophages in humanized TM I/II B C4/8 mice, while its expression is absent from the B cells and macrophages of control mice (FIGS. 7A and 7B). Similarly, humanized MHC I and II are present on the surface of both B cells and macrophages of humanized TM I/II B C4/8 mice (FIGS. 7C and 7D) whereas mouse MHC class I and II molecules were undetectable (FIGS. 7E and 7F). Humanized CD4, CD8 α and CD8β are expressed on the surface of CD3+ thymic cells obtained from humanized TM I/II B C4/8 mice while absent from CD3+ thymic cells in the control mice (FIG. 7G). Humanized TM I/II B CD4/8 express regulatory T cells (Treg) (FIG. 8), NK cells (CD335⁺CD3⁻) and monocytes (CD11b⁺) (data not shown).

Expression in the spleen is depicted in FIGS. 9A-D, and 10A-10G. Spleens of mice humanized for cellular immune system components (TM I/II B CD4/8) comprised comparable absolute numbers of CD3+ cells, and nearly normal proportion of B and T cells (FIG. 9A and data not shown). The frequency and number of F4/80 cells in the spleens of TM I/II B C4/8 mice were compared to control mice (FIG. 9B and data not shown). Mice humanized for cellular immune system components (TM I/II B CD4/8) expressed humanized CD4 and CD8α on CD3+ splenic cells (FIG. 9C). Humanized TM I/II B CD4/8 mice comprised memory effector (CD44⁺CD62L⁻) CD4⁺ and CD8⁺ T cells and central memory (CD44⁺CD62L⁺) CD8⁺ T cells (FIGS. 9D and 9E).

As depicted in FIGS. 10A and 10B, humanized β2M is expressed on the surface of B cells and macrophages in the spleen of humanized TM I/II B C4/B mice, while its expression, and the expression of mouse MHC molecules, are absent from the B cells and macrophages in the spleen of control mice. Similarly, humanized MHC I and II are present on the surface of both B cells and macrophages in the spleen of humanized TM I/II B C4/B mice (FIGS. 10C and 10D) whereas mouse MHC class I and II molecules were undetectable (FIGS. 10E and 10F). Humanized CD4, CD8 α and CD8β are expressed on the surface of CD3+ splenic cells obtained from humanized TM I/II B C4/8 mice while absent from CD3+ splenic cells in the control mice (FIG. 10G). TM I/II B C4/8 mice have near normal expression of splenic regulatory T cells compared to control mice (FIG. 11), and express splenic NK cells (CD335⁺CD3⁻) and monocytes (CD11b⁺).

Example 6: Evaluation of Presentation to and Activation of T Cells with Human Peptide

To determine whether the mice comprising humanized cellular immune system components exhibited humanized T cell immune responses, the ability of splenocytes from mice humanized for cellular immune system components (TM I/II B CD4/8) to present and respond to MAGE-A3, a peptide presented specifically by human HLA-A2, was tested.

MAGE-A3, a peptide presented specifically by human HLA-A2, is synthesized (Celtek Biosciences), diluted in PBS, and mixed in equal volume with Complete Freund's Adjuvant (CFA; Chondrex, Inc.) such that 200 μg of the MAGE-A3 is contained in the 200 μl emulsion. 50 μl of emulsion is injected into 4 spots on each animal. Two spots are each in a hind flank and 2 spots each are near each shoulder of mice homozygous for humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B CD4/8) or control mice which express endogenous MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M.

Spleen suspensions from immunized mice are obtained and dissociated. Red blood cells are lysed in ACK lysis buffer (Life Technologies), and splenocytes are suspended in RPMI complete media. 2×10⁵ of isolated splenocytes in the absence or in the presence of 10 μg/mL or 1 μg/mL of diluted MAGE-A3 peptide are tested per well of PVDF plates (Millipore) coated with 5 μg/mL of the mouse IFN-γ capture antibody (BD Biosciences) in an ELISPOT assay. After a 16-20 hour incubation with peptide, the plates are washed and incubated with biotinylated detection antibody (BD Biosciences), washed, treated with Streptavidin-HRP (MabTech), washed and developed with TMB substrate (Mabtech), and counted by AID Elispot reader.

While only one mouse per genotype is shown, several mice of each genotype were tested, and all samples were run in triplicate with standard deviation shown by error bars. As shown in FIG. 12A, only samples from mice homozygous for each of humanized MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M (TM I/II B CD4/8) responded by secreting IFN-γ after treatment with HLA-A2-specific peptide MAGE-A3, indicating that T cells from these mice were activated after presentation of MAGE-A3 by humanized HLA-A2.

To test the feasibility of isolating human TCRs with therapeutic value from TM I/II B C4/8 mice, the mice were immunized with human tumor associated antigens (TAA). HLA-A2-restricted peptides derived from cancer testis antigens NY-ESO-1 and melanoma-associated antigen (MAGE)-A3 elicited a robust antigen-specific response detectable by IFN-γ production from ex vivo, peptide-pulsed splenocytes (FIGS. 12B-12C). Single NY-ESO-1₁₅₇₋₁₆₅ HLA-A2 tetramer positive T cells from immunized TM I/II B C4/8 mice were sorted, and their TCR alpha and beta variable regions sequenced. TRA and TRB variable cDNA sequences were cloned into lentiviral vectors with fully human TCR constant domains and used to transduce a Jurkat based reporter cell line developed to test class-I restricted antigens (FIG. 12D). TM I/II B C4/8-derived TCRs expressed efficiently in Jurkat cells (FIG. 12E), and mediated robust activation in response to APCs loaded with the NY-ESO-1₁₅₇₋₁₆₅ peptide (FIG. 12F). This response was antigen specific, as transduced Jurkat cells showed no response to control peptides derived from the human transcriptome identified as potential off-target antigens (FIG. 12G).

To test if TCRs isolated from TM I/II B C4/8 mice can mediate a functional anti-tumor response, primary human T cells were engineered to express candidate TCRs by Crispr mediated knockin to the endogenous TRAC locus, a robust strategy for experimental validation that bypasses TCR chain mispairing (FIGS. 12H-12I). T cells expressing NY-ESO-1 specific TCR050 showed dose-dependent in vitro killing of A375 cells, an HLA-A2+ melanoma line that expresses NY-ESO-1, while untransduced T cells expressing polyclonal endogenous TCRs showed none (FIGS. 12J-12L). Cells engineered with an irrelevant TCR against a human papilloma virus (HPV) derived antigen showed some killing activity toward A375, indicating potential reactivity to unknown antigen(s) in A375 cells. However, when these engineered cells were administered to immunodeficient (NSG) mice bearing xenografted A375 tumors, only TCR050 delayed tumor outgrowth (p<0.0001 on day 26 compared to control TCR-treated animals; FIGS. 12M-12O). Moreover, TCR050 continued to blunt tumor growth beyond 40 days after treatment, with 2 of 5 animals remaining tumor-free through day 60, suggesting that the engineered effector T cells are able to persist in vivo. As a correlate of antigen-specific effector T cell activity, serum levels of human IFN-γ 3 days after T cell administration was measured (FIG. 12O). Only TCR050 T cells released significant IFN-γ in each treated mouse, indicating antigen-specific T cell-activation and probable effector function. Together, these data demonstrate the power of the TM I/II B C4/8 technology to generate potent therapeutic TCRs against tumor-specific antigens.

Example 6.2: Materials and Methods Example 6.2.1: Peptide Immunization and Isolation of Antigen Specific TCRS

TM I/II B C4/8 mice were immunized with HLA-A2-restricted peptides derived from human tumor antigens NY-ESO-157-165 (SLLMWITQC; SEQ ID NO:96) and MAGE-A3271-279 (FLWGPRALV; SEQ ID NO:97). 200 μl CFA-emulsified peptide (1 mg/ml) was injected subcutaneously, with 50 ml at each of four sites on the back. At day 14 after injection, the presence of antigen specific T cells was tested by culturing pooled splenocytes and lymph nodes in the presence of 10 μg/ml or no peptide, and quantifying IFN-γ producing cells by ELISpot (BD 551881).

To isolate NY-ESO-1 specific TCRs, splenocytes were isolated 14 days post-immunization and stained with flow antibodies and pMHC tetramer (MBL). Single tetramer-positive T cells were sorted into wells, and TCRA and TCRB sequences were amplified by RT-PCR using 5′ degenerate primer mixes specific for leader sequences, and 3′ primers within the mouse TRAC or TRBC1/2 regions, respectively. Amplicons were cloned into expression vectors in frame with corresponding alpha and beta constant sequences.

Example 6.2.2: TCR Activation Assays in JRT3-T3.5 Cells

TCR activation reporter assays were done in JRT3-T3.5 cells (ATCC TIB-153), a Jurkat subline selected for loss of endogenous TCR surface expression after random mutagenesis (44). A parental JRT3 line for TCR activation assays was established by lentiviral transduction of human CD8A, CD8B, and CD28 constructs along with an AP1 response element (AP1-RE) driven luciferase reporter (Qiagen #CLS-011L). TCRA and TCRB sequences were then introduced by lentiviral transduction, and surface TCR+CD8⁺CD28+ cells were FACS-sorted.

For activation assays, titrated numbers of antigen presenting cells (293T cells expressing human CD80 and CD86) were pulsed with 100 μM NY-ESO-157-165 (SLLMWITQC; SEQ ID NO:96) or control peptides for 2 hours at 37° C., followed by addition of 5e4 TCR-transduced or parental JRT3 cells. After 4 hour's co-culture, luciferase output was measured using OneGlo reagent (Promega E6110) and SpectraMax plate reader (Molecular Devices).

Example 6.2.3: Lentiviral Vectors and Transduction of JRT3 Cells

Lentiviral constructs were generated in pLVX.EF1a.IRES.puro vector (Takara 631988), or related vectors with different antibiotic selection markers. Lentivirus was generated by co-transfection of pLVX vector constructs along with ones for Gag/Pol and VSV-G envelope expression into HEK-293T cells (ATCC CRL-3216) using Lipofectamine LTX reagent (Life Technologies 15338100). Supernatants were harvested 48 hours post-transfection, and lentivirus was concentrated using the Lenti-X reagent (Takara 631232). JRT3 cells were transduced by adding 20 μl concentrated viral supernatant to 2×105 cells in 200 μl total media volume in round-bottom 96-well plates, and spinning plates at 2500 rpm for 90 minutes at RT. Transduced cells were enriched by antibiotic selection, then FACS sorted on the basis of surface marker expression.

Example 6.2.4: Prediction of Potential Off-Target Peptides

Potential off-target peptides are defined as those with predicted affinity for HLA-A2 and high sequence homology to the cognate target peptide. To identify potential off-targets for NY-ESO-157-165 (SLLMWITQC; SEQ ID NO:96), 9-mers from each canonical human protein sequence in the UniProtKB database were extracted and computed predicted binding affinities to HLA-A2, identifying 338,452 peptides with predicted affinity <500 nM. Peptides were filtered for homology to the target peptide at 5 or more amino acid positions and for evidence for expression in essential, normal tissues, narrowing to 26 potential off-target peptides. Four peptides (1 with sequence homology at 6 positions, and 3 of 25 with homology at 5 positions) were selected to test for cross reactivity to isolated NY-ESO-1 specific TCRs.

Example 6.2.5: TCR Knockin to TRAC Locus in Human Primary T Cells

Total T cells were isolated from leukopaks drawn from healthy human donors by negative selection (Stem Cell Technologies #17951). T cells were activated with CD3/CD28 Dynabeads (Life Technologies 11132D) at a 1:1 bead:cell ratio in supplemented CTS OpTmizer (Life Technologies A1048501) media containing 4 mM glutamine, 10 mg/ml gentamicin, 100 U/mL hIL-2 (Miltenyi 130-097-748), and 10 ng/ml hIL-15 (Miltenyi 130-095-765).

The days after activation, beads were removed and cells were nucleofected with Crispr RNP consisting of Cas9 protein (Life Tech A36499) complexed with a mixture of modified synthetic guide RNAs (sgRNAs, IDT) targeting the TRAC (GCUGGUACACGGCAGGGUCA; SEQ ID NO:98) and TRBC1/2 (UGGGAAGGAGGUGCACAGUG; SEQ ID NO:99) genes in their first exons. For each nucleofection, 5e6 T cells were re-suspended in 100 ml nucleofection buffer (Lonza VPA-1002) containing 30 μg total Cas9 protein complexed with 150 pmol of each sgRNA, and electroporated with the pre-installed T-020 pulse program on the Lonza Nucleofector IIb device. Immediately after nucleofection, cells were transferred into equilibrated media, and transduced with adeno-associated virus (AAV, 2e5 viral genomes/cell) vectors encoding homology directed repair (HDR) templates facilitating knock-in of single chain TCRb-F2A-TCRa to the endogenous TRAC locus (FIG. 12H). T cell growth and viability was checked every 2-3 days, and cells were diluted to 0.5-1e6 cell/ml in media with fresh cytokines. TCR knock-in was evaluated by flow analysis with peptide MHC tetramer (HLA-A*0201/NY-ESO-1₁₅₇₋₁₆₅, MBL TB-M011-2) and VR specific antibodies.

Example 6.2.6: Calcein Release Cytotoxicity Assay

A375 (ATCC CRL-1619) target cells were labeled with 8 mM Calcein-AM dye (Life Technologies C1430) in supplemented T cell media for 30 minutes at 37 C, then washed twice in media. 1e4 target cells were plated per well, and T cells were added over a range of dilutions. Wells with labeled target cells but no T cells were included to measure spontaneous release (SR) of dye. To measure maximum release (MR) of dye, 0.5% Triton-X-100 was added to wells containing labeled target cells. After 2.5 hours incubation, assays plates were spun for 5 minutes at 1000 rpm, and 100 ml supernatant was transferred to black, clear bottom 96-well plates. Fluorescence (480 em/530 ex) was read on a SpectraMax plate reader. Percent cytotoxicity was calculated as ((Calcein signal−SR)/(MR−SR))*100.

Example 6.2.7: In Vivo Tumor Experiments

NSG mice (Jackson Laboratory 005557, 7 to 9 weeks-old) were implanted with 5e6 A375 tumor cells subcutaneously on the right flank. T cells were administered intravenously via tail vein on the same day as tumor implant. Tumor volume was measured twice weekly using calipers and calculated by the formula: volume=(length×width²)/2. Blood was collected by submandibular bleed. Serum human IFNγ levels were quantified using the V-Plex Human Pro-inflammatory kit (MesoScale Discovery K15409D), following manufacturer's protocol.

Example 7: Evaluation of T Cell Function Using LCMV Infection Model

To determine whether the mice comprising humanized cellular immune system components exhibited normal response to infection, the ability of humanized mice to clear lymphocytic choriomeningitis virus (LCMV) was tested. LCMV is a mouse tropic virus, where the fate of infection depends on the viral strain. Exposure to Armstrong strain results in an acute infection, where mice can quickly mount a T cell response against the virus and clear the infection in about a week. On the other hand, Clone 13 virus cannot be cleared, and T cells become “exhausted” (expressing markers associated with T cell exhaustion, e.g., PD1, Lag3, Tim3) and chronic infection is established. It has been shown that infection of CD8 depleted or MHC class I deficient mice with Armstrong strain results in maintenance of high viral titers (J. Virol. 68:8056-63 (1994)). Thus, since viral infection depends on T cell activity, LCMV is an ideal model to test for T cell function.

To determine if mice comprising humanized cellular immune system components, e.g., MHC I, MHC II α and β, TCRα and β, CD4, CD8α and β, and β2M, exhibit normal T cell function, both control and humanized (TM I/II B C4/8) mice were infected with 2×10⁵ ffu of Armstrong virus strain i.p. on Day 0. On Days 3, 6, 9, and 12, organs were harvested and viral titers were measured. As shown in FIG. 13A, both control and humanized mice were able to clear Armstrong infection.

Both control and humanized mice were also infected with 4.5×10⁵ ffu of Clone 13 virus i.v. on Day 0, and on Day 21 organs were harvested and viral titers measured. As depicted in FIG. 13B, both mouse strains were able to establish chronic LCMV infection. The ability of humanized mice to express PD1, Lag3, and Tim3, markers of T cell exhaustion, was also measured. Blood was taken from uninfected mice and infected humanized mice 3 weeks post-infection and stained using flow cytometry with PE-Cy7 conjugated anti-PD1 antibody (BIOLEGEND), PerCpCy5.5 conjugated Lag3 antibody (BIOLEGEND), and PE conjugated Tim3 antibody (R&D Systems). Data in FIG. 13C is a quantification of cells staining positive for the indicated receptors. Both humanized (TM I/II B C4/8) mice and control B6 mice expressed all three markers of T cell exhaustion 3 weeks after infection with chronic LCMV Clone 13 strain.

To evaluate memory T cell responses in mice humanized for cellular immune system components, 5 control and 4 humanized mice were infected with 2×10⁵ ffu of Armstrong strain, and on Day 17 super-infected with 4.5×10⁵ ffu Clone 13 strain (2 of each humanized and control mice were mock-infected as an additional control). On Day 31 post initial infection, organs were harvested and viral titers were analyzed. As depicted in FIG. 14, 5/5 control mice and 3/4 humanized mice that have encountered an acute LCMV infection were subsequently protected from chronic LCMV infection, demonstrating intact memory T cell responses in these animals.

To analyze the nature of the cellular responses, control and humanized mice were infected on Day 0 with 2×10⁵ ffu of Armstrong virus strain. On Day 10 (FIGS. 15A-B) or at the indicated time points post infection (FIGS. 15C-D) the specificity of the cellular response was analyzed using three HLA-A2 restricted peptides known to activate human CD8⁺ T cells (GPC10-18, N69-77 or Z49-58), see Botten et al. (2007) J. Virol. 81:2307-17, or gp33, an immunodominant LCMV peptide recognized by mice on a H-2D^(b) background. Specifically, CD8⁺ T cells were isolated from harvested spleens and pulsed with the peptides. CD8⁺ cells producing interferon-γ (IFNγ) were measured by ELISpot (FIGS. 15A-B) or by staining for intracellular IFNγ (FIGS. 15C-D).

CD8⁺ T cells isolated from control animals are specifically activated by the gp33 peptide (FIG. 15A), while CD8+ T cells isolated from humanized animals are activated by the HLA-A2 restricted peptides (FIG. 15B). The time course of CD8+ T cell activation, as monitored by their ability to express IFNγ when stimulated with the peptides, shows in both control and humanized mice CD8+ T cells expand during the first two weeks post infection and are undetectable after the virus is cleared (FIGS. 15C-D). Although the response to gp33 peptide appeared stronger in control animals, it should be noted that gp33 is a known immunodominant LCMV epitope while the immunodominant HLA-A2 restricted LCMV epitope has not been identified. In conclusion, animals comprising a humanized, or substantially humanized T cell immune system are capable of processing LCMV expressed protein, presenting them on humanized MHC molecules and activating T cells via a humanized T cell receptor.

Example 8: Evaluation of I/II B C4/8 Mice Comprising Humanized TCRBDJ1 and TCRBDJ2 Clusters Comprising Murine TCRBDJ1 and TCRBDJ2 Non Coding Sequences and Human TCRBDJ1 and TCRBDJ2 Coding Sequences

Throughout this example 8 and associated figures, TM I/II B C4/8 mice may also be referred to as VelociT mice. Such TM I/II B C4/8 9 (“VelociT” mice) bear nine humanized genes at seven chromosomal loci, which include TCRB murine non-coding sequences at the TCRBDJ1 and TCRBDJ2 clusters.

Example 8.1: Normal Adaptive Immune Cell Development

To ensure this extensive engineering did not interfere with normal immune system development, thorough immunophenotyping of naïve TM I/II B C4/8 mice compared to wild-type (WT) controls with comparable strain background was performed. Flow cytometry analyses showed that numbers and proportions of total T cells, CD4+ helper, and CD8+ cytotoxic T cells, were comparable in spleens of TM I/II B C4/8 and WT mice (FIGS. 16A-B, FIG. 17A). TM I/II B C4/8 mice had proportions and total numbers of splenic B cells (FIGS. 16A and 17A) and serum IgG and IgM (FIG. 17B) comparable to WT controls, indicating a normal B cell compartment. Splenic T cells showed largely normal phenotypically naïve and memory subsets, though with modestly decreased proportions of CD62L-hi CD44-lo naïve CD4+ and CD8+ cells in TM I/II B C4/8 versus WT mice (FIG. 17C).

T cell development in TM I/II B C4/8 mice was normal, showing similar overall numbers and distributions of double negative (DN), double positive (DP), and single positive (SP) CD4+ and CD8+ staged thymocytes to WT mice (FIGS. 16C-D). Established DN subsets delineated by CD44 and CD25 expression were present in TM I/II B C4/8 mice at levels similar to WT, further indicating normal T cell development in TM I/II B C4/8 mice, with intact positive and negative selection on humanized MHC-I and MHC-II. Foxp3+ regulatory T cells (Tregs) were present in TM I/II B C4/8 thymus and spleen, at modestly elevated proportions relative to WT mice (FIGS. 18A and 18B). Collectively, these results indicate a largely normal lymphocyte compartment in naïve TM I/II B C4/8 mice, with proper expression and regulation of humanized TRA, TRB, CD4, and CD8 genes, and development of conventional and regulatory T cell subsets during thymic selection on human MHC.

Example 8.2: Normal Innate Immune Cell Development in TM I/II B C4/8 Mice

TM I/II B C4/8 mice were also analyzed for immune cells responsible for innate immunity and antigen presentation. Natural killer (NK) cells and NK T cells were present at comparable numbers to WT mice (FIGS. 19A and 19B). In addition, neutrophils, monocytes, macrophages, and dendritic cells, including conventional DC1 and DC2 subsets, were present in normal proportions and total numbers in spleen (FIGS. 20A and 20D). Humanization and proper expression of MHC class I and class II molecules were confirmed on professional APCs (FIG. 20B). In addition, expression of human MHC class I was confirmed on non-hematopoietic epithelial cells isolated from kidney, and showed expected upregulation when treated with interferon-γ (IFN-γ FIG. 20C). These data indicate normal development of myeloid lineages and proper expression and regulation of humanized MHC loci in TM I/II B C4/8 mice.

Example 8.3: A Diverse TCR Repertoire in TM I/II B C4/8 Mice

Evidence of proper T cell development in thymus, and the presence of expected phenotypic subsets in the periphery, indicate proper recombination and expression of TCR genes in TM I/II B C4/8 mice. To examine this directly, high-throughput repertoire sequencing of TCR cDNA libraries from splenic naïve CD4⁺ T cells was performed. TM I/II B C4/8 mice utilized diverse TRBV and TRBJ genes, including both the DJC1 and DJC2 gene clusters, except for J2-6, for which the mouse counterpart is a pseudogene (FIGS. 21A-B). TRB V20-1, V28, and V5-1 were the three most frequently used genes, corresponding to previously published reports on repertoire bias in humans. Several distal TRAV genes were detected but at low levels, which is consistent with some studies in human PBMCs (FIGS. 21C-D). Non-template encoded nucleotide additions were observed at both V-D and D-J junctions of TRB transcripts, indicating proper function of mouse terminal deoxynucleotidyl transferase (TdT) with the human segments in TM I/II B C4/8 mice. These results suggest a highly diverse humanized TCR repertoire in TM I/II B C4/8 mice.

Example 8.4: Antiviral CD8 T Cell Responses in TM I/II B C4/8 Mice

To examine T cell responses in TM I/II B C4/8 mice, they were tested with the Lymphocytic Choriomeningitis Virus (LCMV) infection model. Infection of C57BL/6 mice with the Armstrong strain (Arm) of LCMV causes an acute infection eliciting a robust CD8⁺ cytotoxic T-lymphocyte (CTL) response. TM I/II B C4/8 mice challenged with LCMV Arm (2×10⁵ FFU, IP) demonstrated a resolution of acute infection similar to control C57BL/6 mice, albeit with slightly delayed kinetics (d12-19 post-infection [p.i.] vs d8-10 in controls) (FIG. 22A). In contrast to the LCMV Arm strain, infection of mice with the Clone13 (CL13) strain of LCMV (˜5×10⁶ FFU, IV) establishes a chronic virus infection with persistent high virus titers months after infection. CL13 infection of TM I/II B C4/8 mice resulted in persistent infection, with high virus titers 3 weeks after infection similar to controls (FIG. 22B). Chronic LCMV infection in WT mice correlates with a loss of effector T cell function (exhaustion), leading to inadequate control of virus. Chronically infected TM I/II B C4/8 mice showed similar upregulation of inhibitory cell surface molecules PD-1, LAG-3 and Tim-3 on CD8⁺ T cells, consistent with T cell dysfunction following continued antigen stimulation (FIG. 22C). Collectively, TM I/II B C4/8 mice responded to both acute and chronic LCMV infection in a manner similar with WT mice. An important property of adaptive immunity is the ability to mount memory responses to previously encountered pathogens that prevent re-infection. Mice that resolve acute LCMV infection generate a memory CD8⁺ T cell response to the virus that can protect against secondary challenge. To test memory T cell response, TM I/II B C4/8 mice were challenged with LCMV Arm, allowed to resolve infection, and then challenged with CL13. Three of five LCMV immunized TM I/II B C4/8 mice showed complete protection from chronic infection upon re-challenge, and a fourth showed partial protection, while no (0/5) naïve TM I/II B C4/8 mice were immune. Thus, TM I/II B C4/8 mice generate T cells in response to primary LCMV infection that are capable of protecting against a secondary challenge (FIG. 22D).

TM I/II B C4/8 mice respond effectively to LCMV challenge utilizing humanized TCRs restricted on human HLA. To examine whether these responses mirror defined human T cell responses to LCMV, the presence of T cells specific for known HLA-A*02:01 restricted epitopes were interrogated. Of the four HLA-A*02:01 restricted LCMV peptides tested (NP₆₉, GPC₁₀, GPC₄₄₇, Z₄₉), TM I/II B C4/8 mice responded strongly to Z49 from the LCMV Z protein and showed diminished responses to the other peptides (FIGS. 23A-B). These T cell responses demonstrate the processing and presentation of human HLA restricted LCMV peptide epitopes in TM I/II B C4/8 mice, and corresponding CTL responses. It should be noted that TM I/II B C4/8 CD8⁺ T cell responses were distributed across multiple epitopes and not as strong as the dominant response observed to GP33 in C57BL/6 mice. This response pattern may contribute to the delayed clearance kinetics following acute infection (FIG. 22A) and incomplete protection from secondary infection in some TM I/II B C4/8 mice (FIG. 22D). Overall, the acute and chronic LCMV infection data validate TM I/II B C4/8 as a functional platform to study human TCR-HLA driven T-cell activation and exhaustion in vivo.

Example 8.5: Modeling Autoimmunity in TM I/II B C4/8 Mice

The humanization of CD4 and MHC-II in TM I/II B C4/8 mice presents a novel system to study CD4+ T cell responses, develop CD4+ T cell-dependent therapies, and potentially, improve modeling of human autoimmune disease in mice. For instance, the HLA-DR2 haplotype engineered into TM I/II B C4/8 mice is associated with increased susceptibility to multiple sclerosis (MS) in humans, but understanding of specific T cell responses remains limited. To test CD4+ T cell responses and to establish the feasibility of modeling autoimmune disease in TM I/II B C4/8 mice, induction of experimental autoimmune encephalomyelitis (EAE), a widely used model of MS, was attempted.

Human and mouse MOG₃₅₋₅₅ peptides differ only by a single residue, and mouse MOG₃₅₋₅₅ has been shown to bind HLA-DR2 and induce experimental autoimmune encephalomyelitis (EAE) in HLA-DR2-humanized mice. Consistent with these prior studies, immunization of TM I/II B C4/8 mice with mouse MOG₃₅₋₅₅ peptide induced EAE with a mean disease onset on day 10 and occurrence of chronic progressive disease without recovery, but in most cases the disease was not fatal (FIG. 24A). The course of EAE disease in TM I/II B C4/8 mice was very similar to C57BL6/J mice analyzed in parallel, suggesting that MOG₃₅₋₅₅ peptide was similarly presented by human (HLA-DR2) and mouse (I-A^(b)) MHC class II molecules and induced similar MOG-specific CD4+ T cell responses. Accordingly, splenocytes harvested from TM I/II B C4/8 and C57BL/6 mice with EAE showed similar numbers of IFN-g and IL-17A producing cells when pulsed with MOG₃₅₋₅₅ peptide ex vivo (FIGS. 24B-C). The observed disease kinetics and manifestation, along with a preserved Th17 component of EAE disease in TM I/II B C4/8 mice, indicates faithful replication of EAE with utilization of human TCRs restricted to human MHC. Collectively these data indicate that TM I/II B C4/8 mice can mount effective CD4⁺ T cell responses to class-II restricted antigens and can serve as models for autoimmune disease.

Example 8.6: Discussion

The ability to express and isolate human antigen receptors from mice provides a powerful platform for therapeutic discovery. Veloclmmune® mice, which harbor precise, megabase replacements of mouse Ig variable genomic regions with their human counterparts was previously described. This approach to generating human antibodies that undergo natural regulation and selection in developing mouse B cells has produced dozens of monoclonal antibodies in clinical development, including several approved for use in humans. TM I/II B C4/8 mice represent a similar opportunity for the discovery of therapeutic human TCRs.

T cell antigen recognition occurs at an immunologic synapse, wherein TCR and dedicated co-receptors engage peptide-MHC on the APC surface. To achieve effective recapitulation of cognate human interactions in mice, all of these components, totaling 9 protein coding genes at 7 distinct chromosomal loci, were humanized. The humanization strategies replaced the extracellular domains of these surface molecules, while retaining mouse transmembrane and intracellular domains. This design is meant to facilitate physiological interactions with downstream signaling pathways in murine T cells. Accordingly, TM I/II B C4/8 mice showed thymic T cell development and peripheral T cell populations that were similar to WT mice in total numbers, as a proportion of hematopoietic cells, and with respect to expression of phenotypic markers. In addition, B cell, NK cell, and myeloid lineages were normal in TM I/II B C4/8 mice, with expected expression patterns of chimeric MHC-I and -II molecules. Finally, high throughput sequencing of the TCR repertoire in naïve TM I/II B C4/8 mice confirmed faithful recombination and expression of humanized TCR genes, utilizing a diverse repertoire of V(D)J segments. Collectively these results confirm successful expression and regulation of humanized loci, and indicate the development of normal adaptive and innate immune compartments.

Functional studies of TM I/II B C4/8 mice confirmed effective CD4 and CD8 T cell responses. In the LCMV infection model, TM I/II B C4/8 mice resolved acute infection, and the majority of mice were protected from secondary challenge with LCMV CL13, indicating effective intact T cell memory. TM I/II B C4/8 mice chronically infected with CL13 exhibited signs of T cell exhaustion comparable to WT mice, with upregulated PD-1, LAG-3 and Tim-3 (FIGS. 22A-D). Interestingly, the LCMV response in TM I/II B C4/8 mice involved multiple epitopes, including a predominant response to Z49, but also broader responses to multiple peptides (NP₆₉, GPC₁₀, GPC₄₄₇; FIGS. 23A-B). In humans with chronic viral infections, the restricted immunodominant T cell repertoire can mask the more effective subdominant T cell subpopulations. The ability to systematically interrogate the full human TCR repertoire in TM I/II B C4/8 mice may therefore uncover otherwise evasive subdominant epitopes with desirable function, for instance ones targeting conserved regions in mutating viruses.

Induction of EAE in TM I/II B C4/8 mice demonstrated functional MHC class II antigen presentation and CD4+ T cell response. A prior study reported that mice with endogenous TCR loci but expressing transgenic HLA-DR2 and no mouse MHC II developed EAE in response to mouse MOG₃₃₋₅₅ immunization. The data herein confirm HLA-DR2 presentation of encephalitogenic antigen, and further show that human TCRs can drive a CD4+ dominant autoimmune disease in TM I/II B C4/8 mice. Analysis of peptide pulsed splenocytes from EAE mice detected significant IL-17 production and hence Th17 skewing, recapitulating this key pathological feature of mouse EAE and human MS. The data also indicate that TM I/II B C4/8 mice can be used to model autoimmunity driven by human TCRs and antigen-MHC.

The development of normal immune cell populations, a diverse T cell repertoire, and effective CD8+ and CD4+ T cell responses in infectious and autoimmune models point to a powerful new system to investigate T cell mediated disease. With both CD4 and CD8 compartments humanized in one mouse model, TM I/II B C4/8 mice are a significant advance over prior efforts to humanize T cell immunity in mice. Previously generated transgenic mice have been described with randomly integrated human TRA and TRB loci and transgenic HLA-A*0201 on a mouse TCR−/− MHC-I−/− background. These mice produce fully human monoclonal TCRs against HLA-A2 restricted human TAA when immunized. However, these mice show significantly reduced proportions of T cells in blood and thymus compared to WT mice, with especially pronounced deficits of CD8 SP thymic cells. A second iteration of these mice expressed human MHC-II HLA-DR4 on a mouse MHC-II−/− background (but retained mouse MHC I). In this case, mice exhibited significantly reduced thymic and peripheral CD4 T cells and elevated proportions of peripheral DN CD4-CD8-CD3+ T cells compared to WT controls. These mice did not bear humanized co-receptors, and so it is possible the observed deficits reflect sub-optimal thymic selection involving interactions of human HLA and TCR with mouse CD4 and CD8. In contrast, TM I/II B C4/8 mice bear humanizations of both the MHC-I and MHC-II systems, and show physiologically balanced thymic and peripheral T cell subsets comparable to WT mice. Although a more detailed phenotyping of the TCR transgenic mice was not reported, TM I/II B C4/8 mice show largely normal proportions, cell numbers, and phenotypic characteristics of all examined immune subsets. This, in combination with performance in CD4 and CD8 driven disease models, indicate an effective immunocompetent model for study of T cell biology and disease.

A major application of the TM I/II B C4/8 mouse will be as a platform for therapeutic TCR discovery. As proof-of-concept, TM I/II B C4/8 mice elicited robust antigen specific T cell responses to immunization with prototypic tumor associated antigens (see, e.g., FIGS. 12B-12C). Antigen reactivity of a cloned panel of NY-ESO-1-specific TCRs identified by single cell sorting of pMHC-tetramer reactive T cells from immunized mice was further validated. As a preliminary test of specificity, a subset of NY-ESO-1 TCRs was confirmed to not cross-react with a panel of potential cross reactive peptides derived from the human transcriptome, which are predicted to bind HLA-A2 and have homology to cognate NY-ESO-1₁₅₇-165 peptide. A validated TCR was targeted to the endogenous TRAC locus with high efficiency in human cells, and mediated killing of HLA-A2*01 NY-ESO-1+A375 melanoma cells in vitro. These cells further exhibited tumor control in vivo in a xenograft model, indicating robust effector function. Collectively, these experiments validate TM I/II B C4/8 mice for de novo discovery of effective and specific TCRs with anti-tumor activity. Robust and diverse antigen-specific responses to a wide variety of tumor and viral antigens has been shown in these animals, indicating that TM I/II B C4/8 is a transformative platform for TCR discovery (data not shown).

The TM I/II B C4/8 mice reported here are a jumping point for further discoveries that could broaden the impact and availability of T cell therapies. For instance, TM I/II B C4/8 can in principle be customized with any set of human HLA molecules for the discovery of antigens and development of cognate TCRs. The latter has proven daunting in patients, but will be greatly enhanced by a TM I/II B C4/8 platform with customizable HLA haplotypes, amenable to immunization with new antigens as they are discovered, including the vast array of neoantigens potentially mineable from genome sequencing data. In conclusion, TM I/II B C4/8 mice provide a valuable and innovative platform to study human CD4+ and CD8+ T-cell mediated immunity in multiple disease models and can be used to generate potent therapeutic TCRs against viral and human tumor associated antigens.

Example 8.7: Materials and Methods Example 8.7.1: Study Design

Studies were designed to develop a comprehensive system to study T cell function and pathologies in mice utilizing human TCRs and antigens. A second objective was to achieve rapid discovery of candidate therapeutic TCRs, which is currently limited. Therefore, key components of T cell immunity—loci encoding TCR, MHC, and co-receptors recognition domains—were replaced with the corresponding human sequences, validated independently, then combined into a single mouse model that replicates the molecular interactions driving T cell development and response in humans. Immunophenotyping was conducted to confirm preserved presence and function of T cells and other immune compartments. Studies with viral infection and autoimmune models were conducted to show that CD8- and CD4-driven immune responses are functional in TM I/II B C4/8 utilizing human TCRs and human HLA restricted antigens. Finally, immunization with human tumor antigens and isolation of antigen specific TCRs was done to demonstrate TM I/II B C4/8 mice as a tool for therapeutic discovery.

All studies were replicated at least twice with similar results. All mouse studies were designed with sufficient samples sizes to ensure conclusions of statistical significance, as informed by prior studies. Animal studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and all procedures were approved by the by the Regeneron Pharmaceuticals Institutional Animal Care and Use Committee (IACUC).

Example 8.7.2: Gene Humanizations in Mice

BAC clones were obtained from Thermo Fisher Scientific and modified by bacterial homologous recombination (BHR) and ligation using VelociGene® technology as previously described. Targeting of ES cells (F1H4) was performed using the VelociGene® method as previously described. Derivation of mice from modified ES cells by either blastocyst or eight-cell morula injection was as previously described in Examples 1-4.

Example 8.7.3: Immunophenotyping of Mice

Immune cells were isolated from dissected lymphoid organs, cleared of red blood cells by hypotonic lysis, and filtered. For flow cytometric analysis, 10⁶ cells were suspended in 25 μl buffer (1×PBS with 2% FBS and 0.09% sodium azide) containing 0.25 μg Fc block (BD 553142) and incubated at 4° C. for 10 minutes. Flow cytometry antibodies diluted in buffer were then added to wells for a final staining volume of 100 μl, and incubated a further 30 minutes at 4° C. Cells were washed twice in FACS buffer, then re-suspended in 200 μl of fixative solution (1×PBS with 2% paraformaldehyde) for 20 minutes. Cells were pelleted and re-suspended in FACS buffer and analyzed on the BD LSRFortessa within 48 hours.

Example 8.7.4: Sequencing and Analysis of T Cell Repertoire in TM I/II B C4/8 Mice

CD4+ T cells were enriched from spleen by negative selection, and naïve cells were further enriched with CD44 microbeads (Miltenyi Biotech). Total RNA was isolated using the RNeasy Plus kit (Qiagen). cDNA was generated with Superscript II (Life Technologies) using a hybrid universal 5′ primer and oligo(dT). TCRA or TCRB libraries were generated by amplifying with the 5′ adapter primer paired with reverse primers within the mouse TRAC (5′-TCAAAGTCGGTGAACAGGCAGAG-3′; SEQ ID NO:100) or TRBC1/2 (5′-GACCTTGGGTGGAGTCACATTTCTC-3′; SEQ ID NO:101) regions. Libraries were further amplified with indexed adaptor primers, and sequenced on the Illumina Miseq (2×300 cycles).

Raw sequencing reads were de-muliplexed and filtered based on quality, length and perfect match to corresponding constant region primer. Overlapping paired-end reads were merged and analyzed using a local installation of IgBLAST (NCBI, v2.2.25+) to align rearranged TCR chain sequences to human germline V and J gene database, and delineate productive and non-productive joins. CDR3 sequences were extracted using International Immunogenetics Information System (IMGT) boundaries.

Example 8.7.5: LCMV Studies

LCMV Armstrong and CL13 virus strains were grown on BHK-1 cells and titered by standard fluorescent focus assay (FFA) on Vero cells. For primary infection virus clearance studies, mice were dosed with LCMV Armstrong (2E5 FFU, IP), sacrificed at the indicated timepoints, and spleens were harvested and frozen for subsequent virus quantitation using FFA. LCMV specific CD8+ T cell responses in mice were assessed at the indicated timepoints by IFN-γ intracellular cytokine staining (ICS) on splenocytes from infected mice. The class-I LCMV peptides tested were H2db specific GP₃₃₋₄₁ and HLA-A2 specific NP₆₉₋₇₇, GPC₁₀₋₁₈, GPC₄₄₇₋₄₅₅, and Z₄₉₋₅₈. Peptides for HLA-A2 CMV PP65₄₉₅₋₅₀₃, H2Kb OVA₂₅₅₋₂₆₄, and H2db HPV16E7₄₉₋₅₇ were used as negative controls.

The protective memory response to LCMV was assessed in mice infected with LCMV Armstrong (2E5 FFU, IP) 17 days prior, when acute infection had cleared. These LCMV immune mice and naïve controls were then infected with LCMV strain CL13 at a dose known to establish chronic infection in naïve mice (5E6 FFU, IV). Fourteen days post LCMV CL13 infection, the spleens were harvested, frozen, and assessed for virus via FFA.

Example 8.7.6: Experimental Autoimmune Encephalomyelitis (EAE) in Mice

EAE was induced in TM I/II B C4/8 or control C57BL/6 mice with mouse MOG₃₃₋₅₅ peptide (MEVGWYRSPFSRVVHLYRNGK; SEQ ID NO:102) emulsified in complete Freund's adjuvant (CFA) at 1 mg/ml, supplied by Hooke Laboratories (cat. no. EK-2110). 200 μl total emulsion was injected subcutaneously, with 50 μl at each of four sites on the back (upper right, lower right, upper left, lower left). At 2 and 24 hours after peptide injection, 100 ng pertussis toxin suspended in 100 μl 1×PBS was injected intraperitoneally. Mice were monitored for 28 days after peptide injection and assigned a clinical EAE score as follows: 0—no symptoms; 1—limp tail; 2—partial hind leg paralysis; 3—total hind leg paralysis; 4-complete hind and partial front leg paralysis; 5—moribund. At day 28 after injection, the presence of MOG₃₃₋₅₅-specific T cells were tested by culturing pooled splenocytes and lymph nodes in the presence of 10 μg/ml MOG₃₃₋₅₅ or vehicle alone, and quantifying IFN-γ (BD 551881) and IL17A (R&D Systems EL421) producing cells by ELISpot according to the manufacturers' protocols.

EQUIVALENTS

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Entire contents of all non-patent documents, patent applications and patents cited throughout this application are incorporated by reference herein in their entirety. 

1. A mouse or an isolated mouse cell comprising: (A) an unrearranged T cell receptor (TCR) α variable region sequence comprising at least one unrearranged human T cell variable region Vα segment and at least one unrearranged human T cell variable region Jα segment operably linked to a mouse TCRα constant gene sequence, optionally at an endogenous mouse TCRα variable gene locus, wherein the unrearranged TCRα variable region sequence comprises a mouse TCRA non-coding sequence, or (B) an unrearranged TCRβ variable region sequence comprising at least one unrearranged human T cell variable region Vβ segment, at least one unrearranged human T cell variable region Dβ segment, and at least one unrearranged human T cell variable region Jβ segment operably linked to a mouse TCRβ constant gene sequence, optionally at an endogenous mouse TCRβ variable gene locus, wherein the unrearranged TCRβ variable region sequence comprises a mouse TCRB non-coding sequence, or (C) (i) an unrearranged T cell receptor (TCR) α variable region sequence comprising at least one unrearranged human T cell variable region Vα segment and at least one unrearranged human T cell variable region Jα segment operably linked to a mouse TCRα constant gene sequence, optionally at an endogenous mouse TCRα variable gene locus, wherein the unrearranged TCRα variable region sequence comprises a mouse TCRA non-coding sequence, and (ii) an unrearranged TCRβ variable region sequence comprising at least one unrearranged human T cell variable region Vβ segment, at least one unrearranged human T cell variable region Dβ segment, and at least one unrearranged human T cell variable region Jβ segment operably linked to a mouse TCRβ constant gene sequence, optionally at an endogenous mouse TCRβ variable gene locus, wherein the unrearranged TCRβ variable region sequence comprises a mouse TCRB non-coding sequence, wherein the unrearranged human T cell variable region segments are capable of rearranging in a T cell to form genes that encode human T cell receptor variable domains that specifically bind an antigen of interest.
 2. The mouse or isolated mouse cell of claim 1, wherein: (A) the at least one unrearranged human T cell variable region Vα segment comprises a repertoire of unrearranged human T cell variable region Vα segments and the at least one unrearranged human T cell variable region Jα segment comprises a repertoire of unrearranged human T cell variable region Jα gene segments, or (B) the at least one unrearranged human T cell variable region Vβ segment comprises a repertoire of unrearranged human Vβ segments, the at least one unrearranged human T cell variable region Dβ segment comprises a repertoire of unrearranged human Dβ segments, and the at least one unrearranged human T cell variable region Jβ segment comprises a repertoire of unrearranged human Jβ gene segments, or (C) (i) the at least one unrearranged human T cell variable region Vα segment comprises a repertoire of unrearranged human T cell variable region Vα segments, the at least one unrearranged human T cell variable region Jα segment comprises a repertoire of unrearranged human T cell variable region Jα gene segments, (ii) the at least one unrearranged human T cell variable region Vβ segment comprises a repertoire of unrearranged human Vβ segments, the at least one unrearranged human T cell variable region Dβ segment comprises an unrearranged human T cell variable region Dβ1 segment and an unrearranged human T cell variable region Dβ2 segment, and the at least one unrearranged human T cell variable region Jβ segment comprises at least one unrearranged human T cell variable region Jβ1 segment and at least one unrearranged human T cell variable region Jβ2 segment, wherein the mouse TCRB non-coding sequence comprises (i) a mouse TCRBD1-TCRBJ1 non-coding nucleic acid sequence between the at least one unrearranged human T cell variable region Dβ1 segment and the at least one unrearranged human T cell variable region Jβ1 segment and (ii) a mouse TCRBD2-TCRBJ2 non-coding nucleic acid sequence between the at least one unrearranged human T cell variable region Dβ2 segment and the at least one unrearranged human T cell variable region Jβ2 segment.
 3. The mouse or isolated mouse cell of claim 1, wherein: (I) the endogenous mouse TCRα variable gene locus comprises a deletion selected from the group consisting of (a) a deletion of all endogenous TCR Vα gene segments, (b) a deletion of all endogenous TCR Jα gene segments, and (c) a combination thereof, or (II) the endogenous mouse TCRβ variable gene locus comprises a deletion selected from the group consisting of (a) a deletion of all endogenous TCR Vβ gene segments, (b) a deletion of all endogenous TCR Dβ gene segments, (c) a deletion of all endogenous TCR Jβ gene segments, and (d) a combination thereof, or (III) the endogenous mouse TCRα variable gene locus comprises a deletion selected from the group consisting of (a) a deletion of all endogenous TCR Vα gene segments, (b) a deletion of all endogenous TCR Jα gene segments, and (c) a combination thereof, and the endogenous mouse TCRβ variable gene locus comprises a deletion selected from the group consisting of (a) a deletion of all endogenous TCR Vβ gene segments, (b) a deletion of all endogenous TCR Dβ gene segments, (c) a deletion of all endogenous TCR Jβ gene segments, and (d) a combination thereof.
 4. The mouse or isolated mouse cell of claim 1, wherein (I) the endogenous mouse TCRα variable gene locus comprises a replacement selected from the group consisting of (a) a replacement of at least one endogenous T cell variable region Vα gene segment with the at least one unrearranged human T cell variable region Vα gene segment, (b) a replacement of at least one endogenous T cell variable region Jα gene segment with the at least one unrearranged human T cell variable region Jα segment, and (c) a combination thereof, or (II) the endogenous mouse TCRβ variable gene locus comprises a replacement selected from the group consisting of (a) a replacement of at least one endogenous T cell variable region Vβ gene segment with the at least one unrearranged human T cell variable region Vβ segment, (b) a replacement of at least one endogenous T cell variable region Dβ gene segment with the at least one unrearranged human T cell variable region Dβ segment, (c) a replacement of at least one endogenous T cell variable region Jβ gene segment with the at least one unrearranged human T cell variable region Jβ segment, and (d) a combination thereof, or (III) the endogenous mouse TCRα variable gene locus comprises a replacement selected from the group consisting of (a) a replacement of at least one endogenous T cell variable region Vα gene segment with the at least one unrearranged human T cell variable region Vα gene segment, (b) a replacement of at least one endogenous T cell variable region Jα gene segment with the at least unrearranged human T cell variable region Jα segment, and (c) a combination thereof, and the endogenous mouse TCRβ variable gene locus comprises a replacement selected from the group consisting of (a) a replacement of at least one endogenous T cell variable region Vβ gene segment with the at least one unrearranged human T cell variable region Vβ segment, (b) a replacement of at least one endogenous T cell variable region Dβ gene segment with the at least one unrearranged human T cell variable region Dβ segment, (c) a replacement of at least one endogenous T cell variable region Jβ gene segment with the at least one unrearranged human T cell variable region Jβ segment, and (d) a combination thereof. 5.-6. (canceled)
 7. The mouse or isolated mouse cell of claim 1, wherein (I) the endogenous mouse TCRα variable gene locus comprises: (a) a replacement of all endogenous T cell variable region Vα gene segments with all unrearranged human T cell variable region Vα gene segments from TRAV1-1 to TRAV41, and (b) a replacement of all endogenous T cell variable region Jα gene segments with all unrearranged human T cell variable region Jα gene segments from TRAJ1 to TRAJ61; and (II) the endogenous mouse TCRβ variable gene locus comprises (a) a replacement of all contiguous endogenous T cell variable region Vβ gene segments with all unrearranged human T cell variable region Vβ gene segments from TRBV1 to TRBV29-1, (b)(i) a replacement of an endogenous tcrbdj1 cluster with a humanized TCRBDJ1 cluster comprising an unrearranged human TCRBD1 segment and (ii) each of an unrearranged human TCRBJ1-1 segment, an unrearranged human TCRBJ1-2 segment, an unrearranged human TCRBJ1-3 segment, an unrearranged human TCRBJ1-4 segment, an unrearranged human TCRBJ1-5 segment, and an unrearranged human TCRBJ1-6 segment, wherein the humanized TCRBDJ1 cluster comprises a mouse TCRBDJ1 non-coding sequence between the unrearranged human TCRBD1 segment and the unrearranged human TCRBJ1-1 segment and a mouse TCRBDJ1 non-coding sequence between any two consecutive unrearranged human TCRBJ1 gene segments, optionally wherein the unrearranged human TCRBD1 and TCRBJ1 gene segments flank the same mouse TCRBDJ1 non-coding sequences as are normally flanked by the corresponding mouse tcrbdj1 gene segments, and (c)(i) a replacement of an endogenous tcrbdj2 cluster with a humanized TCRBDJ2 cluster comprising an unrearranged human TCRBD2 segment and (ii) each of an unrearranged human TCRBJ2-1 segment, an unrearranged human TCRBJ2-2 segment, an unrearranged human TCRBJ2-3 segment, an unrearranged human TCRBJ2-4 segment, an unrearranged human TCRBJ2-5 segment, an unrearranged human TCRBJ2-6 segment, and an unrearranged human TCRBJ2-7 segment, wherein the humanized TCRBDJ2 cluster comprises a mouse TCRBDJ2 non-coding sequence between the unrearranged human TCRBD2 segment and any unrearranged human TCRBJ2 segment and a mouse TCRBDJ2 non-coding sequence between any two consecutive unrearranged human TCRBJ2 gene segments, optionally wherein the unrearranged human TCRBD2 and TCRBJ2 gene segments flank the same mouse TCRBDJ2 non-coding sequences as are normally flanked by the corresponding mouse tcrbdj2 gene segments. 8.-9. (canceled)
 10. The mouse of claim 1, wherein the mouse expresses a T cell receptor on the surface of a T cell, the T cell receptor comprising a humanized TCRα chain and a humanized TCRβ chain, wherein the humanized TCRα chain is encoded by a rearranged human Vα/Jα sequence operably linked to the mouse TCRα constant region sequence, wherein the rearranged human Vα/Jα sequence is formed by rearrangement of the at least one human T cell variable region Vα segment and at least one human T cell variable region Jα segment, wherein the humanized TCRβ chain is encoded by a rearranged human Vβ/Dβ/Jβ sequence operably linked to the mouse TCRβ constant region sequence, wherein the rearranged human Vβ/Dβ/Jβ sequence is formed by rearrangement of the at least one human T cell variable region Vβ segment, at least one T cell variable region Dβ segment, and at least one human T cell variable region Jβ segment, optionally wherein the TCRβ chain is encoded by a rearranged Vβ/Dβ2/β2 sequence.
 11. The mouse of claim 1, wherein at least 10% of the TCR expressed by the mouse is derived from gene segments from the TCRBDJ1 cluster and at least 10% of the TCR expressed by the mouse is derived from gene segments from the TCRBDJ2 cluster. 12.-29. (canceled)
 30. A genetically modified mouse or isolated mouse cell comprising in its genome: (a) a first nucleotide sequence encoding a chimeric human/mouse CD4 co-receptor that comprises D1, D2 and D3 domains of a human CD4 polypeptide operably linked to D4, transmembrane and cytoplasmic domains of a mouse CD4 polypeptide; (b) a second nucleotide sequence encoding a chimeric human/mouse CD8α polypeptide and a third nucleotide sequence encoding a chimeric human/mouse CD8β polypeptide, wherein the chimeric human/mouse CD8α polypeptide comprises an IgV-like domain of a human CD8α polypeptide operably linked to transmembrane and cytoplasmic domains of an endogenous mouse CD8α polypeptide and wherein the chimeric human/mouse CD8β polypeptide comprises an IgV-like domain of a human CD8β polypeptide operably linked to transmembrane and cytoplasmic domains of an endogenous mouse CD8β polypeptide; (c) a first nucleic acid sequence encoding a chimeric human/mouse MHC II α polypeptide and a second nucleic acid sequence encoding a chimeric human/mouse MHC II β polypeptide, wherein the chimeric human/mouse MHC II α polypeptide comprises α1 and α2 domains of a human HLA class II α polypeptide operably linked to transmembrane and cytoplasmic domains of an endogenous mouse MHC II α polypeptide and wherein the chimeric human/mouse MHC II β polypeptide comprises β1 and β2 domains of a human HLA class II β polypeptide operably linked to transmembrane and cytoplasmic domains of an endogenous mouse MHC II β polypeptide; (d) a third nucleic acid sequence encoding a chimeric human/mouse MHC I polypeptide comprising α1, α2, and α3 domains of a human HLA class I polypeptide operably linked to transmembrane and cytoplasmic domains of an endogenous mouse MHC class I polypeptide; (e) an unrearranged human TCRα variable region sequence comprising at least one unrearranged human T cell variable region Vα segment and at least one unrearranged human T cell variable region Jα segment operably linked to a mouse TCRα constant region sequence; and an unrearranged TCRβ variable region sequence comprising at least one unrearranged human T cell variable region Vβ segment, at least one unrearranged human T cell variable region Dβ segment, and at least one unrearranged human T cell variable region Jβ segment operably linked to a mouse TCRβ constant region sequence, wherein the unrearranged TCRβ variable region sequence comprises a mouse TCRB non-coding sequence; and (f) a polynucleotide encoding a human or humanized β2 microglobulin polypeptide and comprising a nucleotide sequence comprising the nucleotide sequence set forth in exon 1 of an endogenous mouse β2 microglobulin gene operably linked to the nucleotide sequence set forth in exon 2, exon 3, and exon 4 of a human β2 microglobulin gene, optionally wherein the mouse expresses: (A) the chimeric human/mouse CD4 co-receptor, (B) a chimeric CD8 co-receptor comprising the chimeric human/mouse CD8α polypeptide and the chimeric human/mouse CD8β polypeptide, (C) a chimeric MHC II complex comprising the chimeric human/mouse MI-IC II α polypeptide and the chimeric human/mouse MHC II β polypeptide, wherein the chimeric MHC II complex is capable of binding the chimeric human/mouse CD4 co-receptor, (D) the chimeric human/mouse MHC I polypeptide, wherein the chimeric MHC I polypeptide is capable of binding the chimeric CD8 co-receptor, (E) a chimeric human/mouse T cell receptor comprising a humanized TCRα chain and a humanized TCRβ chain, on the surface of a T cell, wherein the humanized TCRα chain is encoded by a rearranged human Vα/Jα sequence operably linked to the mouse TCRα constant region sequence, wherein the rearranged human Vα/Jα sequence is formed by rearrangement of the unrearranged human TCRα variable region sequence comprising the at least one human Vα segment and the at least one human Jα segment, wherein the humanized TCRβ chain is encoded by a rearranged human Vβ/Dβ/Jβ sequence operably linked to the mouse TCRβ constant region sequence, wherein the rearranged human Vβ/Dβ/Jβ sequence is formed by rearrangement of the unrearranged human TCRβ variable region comprising the at least one human Vβ segment, at least one Dβ segment, and at least one human Jβ segment, optionally wherein the humanized TCRβ chain is encoded by a rearranged human Vβ/Dβ2/Jβ2 sequence operably linked to the mouse TCRβ constant region sequence, and (F) the human or humanized β2 microglobulin polypeptide.
 31. The mouse of claim 30, wherein at least 10% of the TCR expressed by the mouse is derived from gene segments from the TCRBDJ1 cluster and at least 10% of the TCR expressed by the mouse is derived from gene segments from the TCRBDJ2 cluster.
 32. (canceled)
 33. A method of making the mouse or mouse cell of claim 1, comprising modifying the genome of the mouse or mouse cell to comprise (a) the unrearranged T cell receptor (TCR) α variable region sequence comprising at least one unrearranged human T cell variable region Vα segment and at least one unrearranged human T cell variable region Jα segment operably linked to a mouse TCRα constant gene sequence, optionally at an endogenous mouse TCRα variable gene locus, wherein the unrearranged TCRα variable region sequence comprises a mouse TCRA non-coding sequence, (b) the unrearranged TCRβ variable region sequence comprising at least one unrearranged human T cell variable region Vβ segment, at least one unrearranged human T cell variable region Dβ segment, and at least one unrearranged human T cell variable region Jβ segment operably linked to a mouse TCRβ constant gene sequence, optionally at an endogenous mouse TCRβ variable gene locus, wherein the unrearranged TCRβ variable region sequence comprises a mouse TCRB non-coding sequence, or (c) both the unrearranged TCRα variable region sequence and the unrearranged TCRβ variable region sequence.
 34. The method of claim 33, wherein the method comprises replacing a contiguous mouse TCRB sequence comprising a mouse TCRBD gene segment and a mouse TCRBJ gene segment with a nucleic acid sequence comprising the at least one unrearranged human T cell variable region Dβ segment, a mouse TCRBD-TCRBJ non-coding nucleic acid sequence, and the at least one unrearranged human T cell variable region Jβ segment, such that the at least one unrearranged human T cell variable region Dβ segment, the mouse TCRBD-TCRBJ non-coding nucleic acid sequence, and the at least one unrearranged human T cell variable region Jβ segment are operably linked to the mouse TCRβ constant gene sequence.
 35. (canceled)
 36. The method of claim 33, further comprising modifying the genome of the mouse or the mouse cell to comprise: (a) a first nucleotide sequence encoding a chimeric human/mouse CD4 co-receptor that comprises D1, D2 and D3 domains of a human CD4 polypeptide and transmembrane and cytoplasmic domains of a mouse CD4 polypeptide; (b) a second nucleotide sequence encoding a chimeric human/mouse CD8α polypeptide and a third nucleotide sequence encoding a chimeric human/mouse CD8β polypeptide, wherein the chimeric human/mouse CD8α polypeptide comprises an IgV-like domain of a human CD8α polypeptide and transmembrane and cytoplasmic domains of a mouse CD8α polypeptide, wherein the chimeric human/mouse CD8β polypeptide comprises an IgV-like domain of a human CD8β polypeptide and transmembrane and cytoplasmic domains of a mouse CD8β polypeptide; (c) a first nucleic acid sequence encoding a chimeric human/mouse MHC II a polypeptide and a second nucleic acid sequence encoding a chimeric human/mouse MHC II β polypeptide, wherein the chimeric human/mouse MHC II a polypeptide comprises α1 and α2 domains of a human HLA class II α polypeptide and transmembrane and cytoplasmic domains of a mouse MHC II α polypeptide, wherein the chimeric human/mouse MHC II β polypeptide comprises β1 and β2 domains of a human HLA class II β polypeptide and transmembrane and cytoplasmic domains of a mouse MHC II β polypeptide; (d) a third nucleic acid sequence encoding a chimeric human/mouse MHC I polypeptide, wherein the chimeric MHC I polypeptide comprises α1, α2, and α3 domains of a human HLA class I polypeptide and transmembrane and cytoplasmic domains of a mouse MHC I polypeptide; and (e) optionally, a β2 microglobulin locus encoding a human or humanized β2 microglobulin polypeptide.
 37. The method of claim 36, wherein the modifying the genome comprises homologous recombination in one or more mouse ES cell(s) such that the first, second, and third nucleotide sequences; the unrearranged human TCRα variable region sequence and unrearranged TCRβ variable region sequence; the first, second, and third nucleic acid sequences; and optionally the β2 microglobulin locus; are added, in any order, into the genome of the one or more mouse ES cell(s).
 38. The method of claim 37, further comprising generating a mouse from the one or more mouse ES cell(s).
 39. A method of obtaining any one of: (1) a TCR protein that is specific for an antigen and comprises a human TCR variable domain, (2) the human TCR variable domain and (3) a nucleic acid sequence encoding the human TCR variable domain, the method comprising isolating from a mouse according to claim 1 any one of: (1) a T cell expressing a TCR protein that is specific for an antigen and comprises both a human TCRα variable domain and a human TCRβ variable domain, (2) either or both (i) the human TCRα variable domain and (ii) the human TCRβ variable domain, and (3) either or both (i) a nucleic acid sequence encoding the human TCR α variable domain and (ii) a nucleic acid sequence encoding the human TCR β variable domain.
 40. The method of claim 39, wherein the method comprises isolating from the mouse a nucleic acid sequence encoding the human TCRα variable domain and a nucleic acid sequence encoding the human TCRβ variable domain, the method further comprising culturing a host cell in sufficient conditions for expressing (i) the nucleic acid sequence encoding the human TCR variable domain in operable linkage with a human TCRα constant region (ii) the nucleic acid sequence encoding the human TCR3 variable domain in operable linkage with a human TCRβ constant region, wherein the nucleic acid sequences encoding the human TCRα variable domain and the human TCRβ variable domain are on the same or different expression vectors. 41.-48. (canceled)
 49. The mouse cell of claim 1, wherein the mouse cell is an embryonic stem cell.
 50. The genetically modified mouse embryonic stem cell of claim 49, comprising in its genome (a) a first nucleotide sequence encoding a chimeric CD4 co-receptor that comprises D1, D2 and D3 domains of a human CD4 polypeptide and transmembrane and cytoplasmic domains of a mouse CD4 polypeptide, (b) a second nucleotide sequence and a third nucleotide sequence respectively encoding a chimeric CD8α polypeptide and a chimeric CD8β polypeptide, wherein the chimeric CD8α polypeptide comprises an IgV-like domain of a human CD8α polypeptide and transmembrane and cytoplasmic domains of a mouse CD8α polypeptide, wherein the chimeric CD8β polypeptide comprises an IgV-like domain of a human CD8β polypeptide and transmembrane and cytoplasmic domains of a mouse CD8β polypeptide, and (c) a first nucleic acid sequence and a second nucleic acid sequence respectively encoding a chimeric MHC II α polypeptide and a chimeric MHC II β polypeptide, wherein the chimeric MHC II α polypeptide comprises α1 and α2 domains of a human HLA class II α polypeptide and transmembrane and cytoplasmic domains of a non-human MHC II α polypeptide, wherein the chimeric MHC II polypeptide comprises β1 and β2 domains of a human HLA class II polypeptide and transmembrane and cytoplasmic domains of a mouse MHC II polypeptide, (d) a third nucleic acid sequence encoding a chimeric MHC I polypeptide, wherein the chimeric MHC I polypeptide comprises α1, α2, and α3 domains of a human HLA class I polypeptide and transmembrane and cytoplasmic domains of a mouse MHC I polypeptide, and (e) an unrearranged human T cell receptor (TCR) α variable region sequence comprising at least one human Vα segment and at least one human Jα segment operably linked to mouse TCRα constant region sequence; and an unrearranged TCRβ variable region sequence comprising at least one human Vβ segment, at least one human Dβ segment, and at least one human Jβ segment operably linked to a mouse TCRβ constant region sequence, wherein the unrearranged TCRβ variable region sequence comprises a mouse TCRB non-coding sequence.
 51. The genetically modified mouse embryonic stem cell of claim 50, wherein (a) the first nucleotide sequence is present at an endogenous CD4 T cell co-receptor locus; (b) the second nucleotide sequence is present at an endogenous CD8α T cell co-receptor locus, and the third nucleotide sequence is present at an endogenous CD8β T cell co-receptor locus; (c) the first nucleic acid sequence is present at an endogenous MHC II α locus and the second nucleic acid sequence is present at an endogenous MHC II β locus; (d) the third nucleic acid sequence is present at an endogenous MHC I locus; and/or (e) the unrearranged human TCRα variable region sequence is present at an endogenous TCRα variable region locus and the unrearranged TCRβ variable region sequence is present at an endogenous TCRβ variable region locus. 52.-53. (canceled)
 54. A mouse embryonic stem (ES) cell made by the method of claim
 33. 55. A targeting vector comprising (1) 5′ and 3′ homology arms for targeting a mouse TCRBDJ region, (2) an unrearranged human TCRBD segment, (3) an unrearranged human TCRBJ segment, and (4) a mouse TRCBDJ non-coding sequence, wherein targeting vector comprises the mouse TCRBDJ non-coding sequence between the unrearranged human TCRBD segment and any unrearranged human TCRBJ gene segment and between any two consecutive unrearranged human TCRBJ gene segments, optionally wherein the unrearranged human TCRBD and TCRBJ gene segments flank the same mouse TCRBDJ non-coding sequences as are normally flanked by the corresponding mouse TCRBDJ gene segments.
 56. (canceled)
 57. A mouse genome or mouse cell comprising the targeting vector of claim
 55. 